Slovak Journal of Animal Science, volume 46, number 1, 2013 by CVŽV Nitra

Number

2013 Volume 46 46 (1) 1-38 ISSN 1337-9984

Slovak Journal of

Animal Science ANIMAL PRODUCTION RESEARCH CENTRE NITRA

Slovak Journal of Animal Science Editorial office

Formerly Journal of Farm Animal Science

Editor-in-chief: Ladislav Hetényi, Animal Production Research Centre Nitra, Slovak Republic Executive editor: Ludmila Hanuliaková, Animal Production Research Centre Nitra, Slovak Republic Technical editor: Marta Vargová, Animal Production Research Centre Nitra, Slovak Republic

Editorial board Daniel Bíro, Slovak University of Agriculture Nitra, Slovakia Zsuzsanna Bosze, Agricultural Biotechnology Center, Gödöllö, Hungary Jan Brouček, Animal Production Research Centre Nitra, Slovakia Jozef Bulla, Slovak University of Agriculture Nitra, Slovakia Ondrej Debrecéni, Slovak University of Agriculture Nitra, Slovakia Andrzej Filistowicz, The Faculty of Biology and Animal Science, University of Enviromental and Life Science, Wroclaw, Poland Roland Grossmann, Institute of Animal Science Mariensee, Germany Jarosław Olav Horbańczuk, Institute of Genetics and Animal Breeding of the Polish Academy of Sciences in Jastrzębiec n/Warsaw, Poland Peter Chrenek, Animal Production Research Centre Nitra, Slovakia Jozef Laurinčík, Constantine the Philosopher University Nitra, Slovakia Juraj Koppel, Institute of Animal Physiology SAS, Košice, Slovakia Alexander Vladimírovič Makarevič��, Animal Production Research Centre Nitra, Slovakia Peter Massanyi, Slovak University of Agriculture Nitra, Slovakia Gábor Mészáros, University of Natural Resouces and Life Sciences, Division of Livestock Sciences, Vienna, Austria Štefan Mihina, Animal Production Research Centre Nitra, Slovakia Shoukhart M.Mitalipov, Oregon Health & Science University, Beaverton, U.S.A. Jaana Peippo, MTT Agrifood Research Finland, Jokioinen, Finland Dana Peškovičová, Animal Production Research Centre Nitra, Slovakia Juraj Pivko, Animal Production Research Centre Nitra, Slovakia Josef Přibyl, Research Institute for Animal Production, Praha – Uhříněves, Czech Republic Ján Rafay, Animal Production Research Centre Nitra, Slovakia Alexander Sirotkin, Animal Production Research Centre Nitra, Slovakia Pavel Suchý, Department of Nutrition, Dietetics, Zoohygiene and Plant Products, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic Milan Šimko, Slovak University of Agriculture Nitra, Slovakia Peter Šútovský, University of Missouri – Columbia, U.S.A. Vladimír Tančin, Animal Production Research Centre Nitra, Slovakia Manuel Tena-Sempere, University of Cordoba, Spain Miroslav Valent, West Virginia University, Morgantown, U.S.A. Igor Valocký, University of Veterinary Medicine, Košice, Slovakia Scientific English: Shubhadeep Roychoudhury, Alexander V. Makarevič

Aims and scope Slovak Journal of Animal Science (ISSN 1337-9984) is an international scientific journal that publishes original scientific papers, reviews, short communications, chronicles of important jubilees, reports of participation in important international conferences on animal science in English language. Topic of the journal are problems of animal production, mainly in the sphere of genetics, breeding, nutrition and feeding, physiological processes of digestion, conservation and treatment of feeds, biotechnology, reproduction, ethology, ecologization of breeding, biology and breeding technology in farm animals, quality of meat, milk, wool, economy of breeding and production of farm animals, mainly: cattle, pigs, sheep, goats, horses, poultry, small farm animals and farm game. There are published also articles from the sphere of biochemistry, genetics, embryology, applied mathematical statistics as well as economy of animal production. There can be published also articles from the sphere of veterinary medicine concerning the themes of the journal. No part of this publication may be reproduced, stored or transmitted in any form without prior written permission of the Publisher.

Online version of the journal (ISSN 1338-0095) is at disposal at http://www.cvzv.sk/index.php/slovak-journal-of-animal-science. Permission of the Publisher is required to store or use electronically any material contained in this journal.

This journal is comprised in: AGRIS/FAO database (the full texts, too); CAB Abstracts; Knovel. Slovak Journal of Animal Science is published under the authorization and direction of the Animal Production Research Centre (APRC) Nitra, Slovak Republic. Editorial office, orders, subscription and distribution: APRC Nitra, Hlohovecká 2, 951 41 Lužianky, Slovak Republic. Phone +421 37 6546 249; e-mail: editor@cvzv.sk; http://www.cvzv.sk/ Filed at the Ministry of Culture of the Slovak Republic: EV 3659/09. © APRC Nitra in Publishing house Publica Nitra, 2013.

Effects of GnRH agonist (CinnaRelin) on Reproductive Performance in Synchronized Iranian Crossbred Ewes during the Breeding Season

A. OLFATI*, GH. MOGHADDAM Department of Animal Science, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

ABSTRACT This study was carried out to investigate the efficiency of GnRH on reproductive performance of synchronized Iranian crossbred ewes during the breeding season. The ewes (n = 90) were treated with CIDR containing 30 mg progesterone for 14 days and were injected with 400 IU PMSG at the time of removal of the CIDR. The ewes were then divided into three equal groups of 30 ewes each. One milliliter of water soluble was administered to each ewe in the Group 1 (control) at the time of both the CIDR withdrawal and AI. In the group 2 approximately 12.5 µg of GnRH (CinnaRelin) were injected to each ewe immediatly after AI. In the 3 Group, 12.5 µg GnRH were injected to each ewe on 11 to 13 days post-insemination. Intracervical AI with diluted fresh semen was performed once at 12 hours following the estrus onset. Lambing and multiple birth rates were significantly higher (P<0.05) in the Group 2 (123.3 % and 45.8 %) in comparison to other groups. Pregnancy and fecundity rates tended to be higher in the Group 2 (80 % and 154.2 %) and the Group 3 (76.6 % and 130.4 %), when compared with the control Group (66.6 % and 110 %). However, the differences among any groups in terms of the number of twin lambs, gestation period, lambs birth weight and male and female lamb rate were statistically insignificant (P>0.05). In conclusion, GnRH administration improved the reproductive performance of the ewes when administrated at the time of AI or during the midluteal phase after AI. Key words: Iranian crossbred ewes; lambing rate; GnRH; synchronization

INTRODUCTION The Arkhar-Merino is a sheep breed which was produced by crossbreeding between wild Arkhar rams with ewes of the Novocaucasian Merino, Précoce and Rambouillet breeds (Ernst and Dmitriev, 2007). The goal of the crossbred sheep production in the northwest Iran was genetic improvement of local wool trait breeds. The most economically important trait in sheep production is reproduction and it can be manipulated using hormonal treatments (Atsan et al., 2007). Hormonal treatment to control ovulation and reproduction is a prerequisite for successful breeding and increasing the number of pregnant females (Husein et al., 2005), resulting in a short breeding period and more uniform newborn crop (Husein and Kridli, 2003). Gonadotrophin releasing hormone

*Correspondence: E-mail: a.olfati65@gmail.com. Ali Olfati, Department of Animal Science, University of Tabriz, �� 5166614766 Tabriz, Iran Tel.: +98 411 3392041 Fax: +98 411 3356004

(GnRH) plays a key role in the reproductive function and has a potential for fertility control in mammals (Diskin et al., 2002). In many studies where GnRH was used, it was aimed to increase the progesterone levels (P4) in serum, to decrease the early embryonic deaths and as a result to increase the pregnancy rates. GnRH injection causes a predictable release of luteinizing hormone (LH) and a significant increase in serum P4 (Stevenson et al., 1993). In addition, it is reported that GnRH injection on 11 to 14 days (during the mid luteal phase) after artificial insemination (AI) increased serum concentrations of P4 and caused a tendency toward higher pregnancy rates in cattle (Hansen, 2002). It is thought that GnRH treatment may increase the chances of embryo survival by improving luteal function and/or interfering with the luteolytic mechanism (Beck et al., 1994; Cam et al.,

Received: August 3, 2012 Accepted: December 5, 2012

Original paper 2002). The application of GnRH reduced the variation in the timing of the LH surge for goats, improving the synchrony of ovulation (Pierson et al., 2003). The GnRH administration at the time of oestrus increased serum concentrations of P4 and improved pregnancy rates in cows (Ullah et al., 1996). Because of decreasing Iranian crossbred ewe population in recent years (Jafari, 2008), the strategies which can improve population of this animal can be profitable for farmers and conserving this animal. This study is the first to report the GnRH treatment on reproductive indices of Arkhar-Merino ewes reared in the northwest Iran. Hence, this study was designed to determine the effects of single administration of GnRH treatment at the time of AI or during the mid luteal phase after AI on their pregnancy rate and lambing performance in Iranian crossbred ewes synchronized with CIDR+PMSG technique during the breeding season.

MATERIAL AND METHODS Hormonal preparations Controlled internal drug release with 30 mg of progesterone, a progestagen analogue (InterAg, Hamilton, New-Zealand), PMSG (folligon; Intervet International B.V., Boxmeer, the Netherlands) and GnRH (each ml of GnRH contains 5µg, CinnaRelin; CinnaGen Biopharma Co, Tehran, Iran) were purchased from Intervet Drug Industry (Tehran, Iran). Animals and location In the present study, a total of 120 clinically healthy, free of reproductive disorders and once lambed adult Iranian crossbred ewes (forty-five Arkhar-Merino× Ghezel (AM-GH); forty-five Arkhar-Merino×Moghani (AM-MG), 2-5 years old, weighing 40-50 kg), and six healthy adult crossbred rams (3-4 years old, weighing 65-75 kg) were used. The study was carried out in breeding season (June/September, 2011) at the agricultural research station, University of Tabriz, East Azerbaijan province at 38°07´N, 46º29´E and altitude of 1567 m. The ewes were kept indoors at night and outdoors most of the day. Indoors, the ewes were fed a concentrated diet based on cottonseed meal, barley and wheat bran having 2450 kcal ME and 14 % crude protein through the experimental period. Water and a mineral supplement was available ad libitum. Treatment schedule The ewes (n = 90) were treated with CIDR containing 30 mg progesterone for 14 days and were injected with 400 IU PMSG at the time of the CIDR removal. The ewes were then divided into three equal groups of 30 ewes each. One milliliter of water soluble was administered to each ewe in the Group 1 (control) at

Slovak J. Anim. Sci., 46, 2013 (1): 1-6 time of both the CIDR withdrawal and AI. Approximately 12.5 µg of GnRH (CinnaRelin) was injected to each ewe in the Group 2 immediatly after AI. In the Group 3, 12.5 µg of GnRH was injected to each ewe on 11 to 13 days post-insemination. In order to determine the time of the estrus onset, all the ewes were monitored every 6 hours from 12 to 96 hours following CIDR withdrawal using six teaser rams equipped with an apron. Semen collection, processing and insemination of the ewes Semen was collected from all the rams with aid of an artificial vagina, the ejaculates were held in a warm water bath at 37°C until their assessment. Each ejaculate was immediately evaluated to determine motility of the semen. Sperm motility was identified as the percentage of sperm cells that demonstrated progressive motility, from 0 to 100 %, by a qualified and experienced investigator. Semen was placed on a heated (37°C) glass slide and scoring was performed at microscopic magnification of 200×. The mean of the two successive estimates was used as the final motility score. Spermatozoa concentration was determined by a hemocytometric method using the standard haemocytometer (Improved Neubauer, Deep 1/10 mm, Labart, Germany) slide and dilution pipette designed for counting red blood cells (Gur et al., 2005). All the ejaculates having forward progressive motility more than 70 %, were then pooled and diluted at the 1 : 1 ratio (semen : diluents) at 37°C with the skim milk extender. Hence, 1000 IU sodium G penicillin and 1000 µg dihydrostreptomycine sulphate was added to 1 ml of the diluted semen. The diluted semen was kept at 37°C in a water bath until insemination. Each ewe was intracervically inseminated at 12 h following the estrus onset using specific insemination catheter containing 0.5 ml of the diluted semen (approximately 2×108 spermatozoa/ml). Pregnancy diagnosis Pregnancy was diagnosed by transabdominal ultrasound examination at day 57 after AI. The ultrasound equipment used was a real-time scanner (Shimasonic, Model SDL-32C; Shimadzu Deutschland GmbH, Duisburg Germany). The probe was lubricated with ultrasonic gel (Ultrasonic Gel, Virbac Australia Pty Ltd, Peakhurst, NSW) and placed in contact with the abdomen in order to visualize the foetus on the screen. The number of ewes carrying at least one live foetus was recorded (Reyna et al., 2007). Statistical analysis Values concerning gestation period and lamb birth weight were compared by one-way analysis of variance (ANOVA) and post hoc Tukey-HSD test (SAS, 2008). The chi-squared test was performed to determine the differences among all groups concerning the other

Slovak J. Anim. Sci., 46, 2013 (1): 1-6

Original paper

reproductive traits measured. Statistical significance was proved at P<0.05. Pregnancy, lambing, and fecundity rates were calculated as follows (Zeleke et al., 2005): pregnancy rate =

ewes lambing × 100 ewes inseminated

lambing rate =

lambs born × 100 ewes inseminated

fecundity rate =

lambs born × 100 ewes lambing

RESULTS AND DISCUSSION The values concerning reproductive parameters, gestation period and lambs birth weight are presented in Table 1. Lambing and multiple birth rates were significantly higher (P<0.05) in Group 2 (123.3 % and 45.8 %) in comparison to Group 3 (100 % and 26 %) and Group 1 (73.33 % and 10 %, control Group). When the number of single lambs was statistically compared only a significant difference (P<0.05) between the Groups 1 (90 %) and 2 (13 %) was recorded. Pregnancy and fecundity rates tended to be higher in the Group 2 (80 % and 154.2 %) and the Group 3 (76.6 % and 130.4 %), when compared with the Group 1 (66.6 % and 110 %). However, the differences among any of the groups in terms of the number of twin

Table 1: Reproductive performance of ewes after different treatment protocols (Group 1: progestagen + PMSG + post-insemination NaCl; Group 2: progestagen + PMSG + insemination GnRH; Group 3: progestagen + PMSG + post (11 to 13 days) – insemination GnRH) Variable 1

Group 2

Number of ewes

Pregnancy rate (%)

Lambing rate (%)

Fecundity rate (%)

110

Multiple birth rates (%)

10 % (2/20)

Number of lambs

Single

18 (90 %)a

13 (54.2 %)b

17 (73.9 %)a

Twin

2 (10 %)

9 (37.5 %)

5 (21.7 %)

Triplet

2 (8.3 %)

1 (4.4 %)

Gestation period (days)

149-157

148-155

149-153

Lambs birth weight (kg)

5.1 ± 0.4

4.3 ± 0.4

4.6 ± 0.4

Female lamb rate (%)

45.4 (10/22)

51.4 (19/37)

50 (15/30)

Male lamb rate (%)

54.6 (12/22)

48.6 (18/37)

50 (15/30)

Data are expressed as mean ± S.M.E. a, b values within a rows having different superscripts differ significantly (P<0.05)

66.6

76.6

73.33b

123.3a

100b

154.2

lambs, gestation period, lamb birth weight and male and female lamb rate were statistically insignificant (P>0.05). Little is known regarding the reproductive indices of Arkhar-Merino rams and ewes under normal environmental conditions in Iran. In the present study in attempt to reduce embryonic morality and hence to improve reproductive performance of the Iranian

130.4

45.8 % (11/24)

26 % (6/23)b

crossbred ewes, a single administration of GnRH (12.5 µg) treatment was applied during the breeding season. The results obtained in this study showed that administration of GnRH at the time of AI and on 11 to 13 days post-insemination (during the mid luteal phase) improves lambing and twinning rates in synchronized ewes. These results are in agreement with the previous

Original paper reports in ewes, where GnRH administration improved reproductive performance (Safranski et al., 1992; Turk et al., 2008). However, some studies have reported that GnRH administration had no effect on reproductive performance in dairy cows (Ryan et al., 1994; Tefera et al., 2001). Differences in the effectiveness of GnRH therapy on reproductive performance reported by others could be related to the genetics, age and different GnRH analogues or their doses. In the present study a significant effect of exogenous GnRH treatment on number of single lambs of ewes was observed in the Groups 1 and 2 (P<0.05). GnRH, used alone, induced a closely synchronized LH surge 2 hours after intramuscular injection during the breeding (Rubianes et al., 1997a). The beneficial effect of GnRH supplementation at time of insemination increases conceptus growth (Kleemann et al., 1994; Lashari and Tasawar, 2007). This would have helped to improve embryo survival as larger conceptuses produce more IFN-tau, thereby more effectively suppressing the luteolytic mechanism and allowing more time for the establishment of pregnancy (Nephew et al., 1994). Reyna et al. (2007) reported that GnRH application had a positive effect in synchronizing the time of ovulation but had no effect on the growth or atresia of the ovulatory or subordinate follicles. Administration of GnRH resulted in a rapid increase in plasma LH concentration and also in an increase in plasma progesterone concentrations in sheep (Beck et al., 1994; Cam et al., 2002). A functional courpus Luteum (CL) is required for the maintenance of pregnancy (Howell et al., 1994). The number of accessory CLs is increased with the administration of GnRH (Beck et al., 1994; Cam et al., 2002). Although plasma LH and P4 concentration and the number of accessory CLs was not determined in the present study, the effect of GnRH on embryo survival in ewes may occur through GnRH-stimulated LH surge stimulating production of progesterone by the CL and/or causing ovulation and the formation of accessory CLs. GnRH also promotes the formation of an accessory CL when injected at dioestrus (Stevenson et al., 1996). Injection of GnRH may have stimulated the transformation of small cells to large cells which had a higher basal secretion rate of P4 (Stevenson et al., 1993; De Rensis and Peters, 1999). The results of the present study showed that GnRH administration on the day of insemination improved rate and number of lambs born. This is in agreement with the previous findings of Peters et al. (1992); Cam et al. (2002); Khan et al. (2007) and Lashari and Tasawar (2007; 2010), who observed positive effect of GnRH administration on the day of mating on embryo survival in sheep and dairy cows. GnRH treatment on the day of insemination can increase pregnancy rate in cows by 6 to 7 % (Peters et al., 1992).

Slovak J. Anim. Sci., 46, 2013 (1): 1-6 During the first 3 weeks of pregnancy, 30-40 % of fertilized eggs are lost in sheep and goats (Nancarrow, 1994; Michels et al., 1998). Of this total loss, 70-80 % occurs between days 8 and 16 after insemination (Sreenan et al., 1996), the majority of this is the result of embryo mortality (Beck et al., 1996). Fertilization failure accounts for only 5-10 % of losses (Wilmut et al., 1986). One of the major causes of embryonic loss is thought to be the inadequate luteal function (Nancarrow, 1994). Results of the present study showed that the treatment of ewes with CinnaRelin (a GnRH analogue) once during the mid luteal phase after AI (on days 11 to 13 days post-insemination) improved pregnancy rates of Iranian crossbred ewes during the breeding season. This is in agreement with the findings of Batavani and Eliasi (2004) and Macmillan and Thatcher (1991) that GnRH administration results in an increase in pregnancy rates in river buffaloes and dairy cattle, respectively, whereas others reported no beneficial effect in cattle (Jubb et al., 1990; Jayakumar and Vahida, 2000). Alacam et al. (1999) studied the effect of gonadorelin (a GnRH analogue) administered on day 12 post-mating in a small number of Angora goats and reported that GnRH administration increased pregnancy rate, kidding rate and litter size. The results of the present study are in agreement with these findings. When GnRH analogue was given on the day 12 after AI in cows, serum progesterone concentration was not affected, while serum LH levels increased significantly compared to controls (Yildiz et al., 2009). The response to GnRH depends on the phase of the cycle at which the hormone is administered (Geary et al., 2000). There was no significant difference in lamb birth weight and gestation period between the GnRH treated and the control groups in the present study. This result is in concert with the trial of Turk et al. (2008) in Awassi ewes during the breeding season. It has been reported that progesterone supplementation increases subsequent fetal growth (Kleemann et al., 1994). Therefore, it is possible that the GnRH administration may also stimulate subsequent fetal growth through enhanced luteal activity. Additionally, no statistically significant differences were observed among any groups in terms of the male and the female lamb rate, which agreed with the result obtained by Turk et al. (2008) in Awassi ewes. The results of the present study demonstrated that administration of exogenous GnRH (12.5 Âľg) significantly affected reproductive performance of ewes when administered at the time of AI or during the mid luteal phase after AI. Increased pregnancy rate and litter size in GnRH administered ewes are thought to be the results of GnRH effect on increasing embryo survival through enhanced luteal function. Additionally, this technique (CIDR+PMCG) is an effective tool for estrus synchronization in Iranian crossbred ewes at the northwest Iran during the breeding season.

Slovak J. Anim. Sci., 46, 2013 (1): 1-6 ACKNOWLEDGMENT The authors are thankful to Dr. H. Daghigh Kia, Dr. N. Pirany, and Dr. S. A. Rafat for their help and collaboration during this work.

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Slovak J. Anim. Sci., 46, 2013 (1): 1-6 SAFRANSKI, T. J. – LAMBERSON, W. R. – KEISLER, D. H. 1992. Use of melengestrol acetate and gonadotropins to induce fertile estrus in seasonally anestrous ewes. J. Anim. Sci., vol. 70, 1992, p. 2935-2941. SAS. (2003). SAS Users Guide. SAS Institute Inc. Cary, NC. SREENAN, J. M. – DISKIN, M. G. – DUNNE, L. 1996. Embryonic mortality: the major cause of reproductive wastage in cattle. In: Proceedings of the 47th Annual Meeting of the European Association of Animal Production, Lillehammer, August 1996. STEVENSON, J. S. – PHATAK, A. P. – RETTMER, I. M. M. O. – STEWART, R. E. 1993. Postinsemination administration of receptal: follicular dynamics, duration of cycle, hormonal responses, and pregnancy rates. J. Dairy Sci., vol. 76, 1993, p. 2536-2547. STEVENSON, J. S. – KOBAYASHI, Y. – SHIPKA, M. P. – RAUCHHOLZ, K. C. 1996. Altering conception of dairy cattle by gonadotropin-releasing hormone preceding luteolysis induced by prostaglandin F2α. J. Dairy Sci., vol. 79, 1996, p. 402-410. TEFERA, M. – CHAFFAUX, S. – THIBIER, M. – HUMBLOT, P. 2001. A short note: Lack of effect of post-AI hCG or GnRH treatment on embryonic mortality in dairy cattle. Livest. Prod. Sci. vol. 71, 2001, p. 277-281. TURK, G. – GUR, S. – SONMEZ, M. – BOZKURT, T. – AKSU, E. H. – AKSOY, H. 2008. Effect of exogenous GnRH at the before time of artificial insemination on reproductive performance of Awassi ewes synchronized with progestagen-PMSG-PGF2alpha combination. Reprod. Dom. Anim., vol. 43, 2008, p. 308-313. ULLAH, G. – FUQUAY, J. W. – KEAWKHONG, T. – CLARK, B. L. – POGUE, D. E. – MURPHEY, E. J. 1996. Effect of gonadotropin-releasing hormone at estrus on subsequent luteal function and fertility in lactating Holsteins during heat stress. J. Dairy Sci. vol. 79, 1996, p. 1950-1953. WILMUT, I. – SALES, D. I. – ASHWORTH, C. J. 1986. Maternal and embryonic factors associated with prenatal loss in mammals. J. Reprod. Fertil., vol. 76, 1986, p. 851-864. ZELEKE, M. – GREYLING, J. P. C. – SCHWALBACH, L. M. J. – MULLER, T. – ERAMUS, J. A. 2005. Effect of PMSG dose on oestrus synchronization and fertility in Dorper ewes during the transition period. Small. Rumin. Res., vol. 56, 2005, p. 47-53.

Comparison of milk quality of transgenic rabbits carrying different gene constructs

P. Chrenek1,2, A. Makarevich1, D. Kozelová2, L. Hiripi3, Zs. Bozse3 Animal Production Research Centre Nitra, Slovak Republic Faculty of Biotechnology and Food Science, Slovak University of Agriculture, Slovak Republic 3 Agriculture Biotechnological Centre, Godollo, Hungary 1 2

ABSTRACT Basic objective of this research was to compare the milk quality of New Zealand White transgenic rabbit females expressing recombinant human protein C (hPC) or recombinant human factor VIII (hFVIII) in the mammary gland during first lactation with that of non-transgenic rabbit females of the same age. Transgenic founders were generated by the microinjection of foreign DNA (mWAP-hPC gene construct or mWAP-hFVIII) into the egg. Then several generations of transgenic rabbits were obtained after mating of transgenic founder rabbits with non-transgenic rabbits. Quality of milk (content of fat, protein, lactose, dry ash, and some minerals) from transgenic rabbits was determined. Our analyses of transgenic rabbit milk samples showed that all the transgenic females (carrying hPC or hFVIII) tested in this work produced lower or higher amounts of recombinant human protein C or recombinant human FVIII, depending on transgenic females, with proved biological activity. No significant differences in the content of milk fat, protein, lactose and somatic cell counts were found. Our results showed that milk performance of transgenic rabbits over several generations (using both gene constructs) did not differ significantly from those of non-transgenic animals. Key words: transgenic rabbit; milk quality; mWAP-hPC and mWAP-hFVIII gene

INTRODUCTION The use of transgenic mammary gland as bioreactors („pharmaceutical farming“) is cost-effective and variability in post-translational modification is an alternative to cell culture methods (Garber, 2000). The mammary gland is the most promising target tissue because it produces large amounts of protein in a temperature-regulated fluid that may be collected daily in a non-invasive fashion. Transgenic animals are not only cost-effective bioreactors, but, with the complex secretory cell types and organs of the mammalian organism, can perform much more complicated protein modifications than simply cultured cells. Transgenic animals used as bioreactors to produce human proteins represent new horizon in animal husbandry often followed by *Correspondence: E-mail: chrenekp@yahoo.com Peter Chrenek, Animal Production Research Centre Nitra, Hlohovecká 2, 951 41 Lužianky, Slovak Republic Tel.: +421 37 6546285 Fax: +421 37 6546189

low viability of newborn animals (Lubon et al., 1996, Chrenek et al., 2007a). Milk is usually the sole source of nourishment of young mammals, therefore offspring growth and development depends on milk. Rabbit milk yield may be affected by breed of doe (Lukafahr et al., 1983), nutrition (Chrastinová et al., 1997), number of kids suckling and their age of weaning (Taranto et al., 2003) and pregnancy during lactation (Lukafahr et al., 1983). Intensive recombinant human protein production by the mammary gland of transgenic rabbit necessitates the knowledge of the lactation curve and quality (composition) of milk as a possible effect of transgenesis on milk yield. The objective of this research was to compare the milk quality of New Zealand White transgenic rabbit females expressing recombinant human protein C Received: January 11, 2013 Accepted: January 28, 2013

Original paper

Slovak J. Anim. Sci., 46, 2013 (1): 7-10

(hPC) or recombinant human factor VIII (hFVIII) in the mammary gland during first lactation with that of nontransgenic rabbit females of the same age.

MATERIAL AND METHODS Biological material Transgenic rabbit females from second generations were obtained after mating of transgenic rabbit females carrying the mWAP-hPC or mWAP-hFVIII transgene (Chrenek et al., 2002, 2005) with non-transgenic males. The animals of about 4-5 months age at 3.5 - 4.0 kg of weight were used in this experiment. All females had from 7 to 9 pups per litter. They were housed in individual steel cages, with adjacent box for a possibility to separate litter during entire experiment, in controlled environment (constant temperature and light-dark regime). Food and water were supplied ad libitum. Rabbit milk analysis Milk samples (10 ml) were collected from each lactating transgenic and non-transgenic females on day 21st using “home made air vacuum pump” from several parts (nipples) of the mammary gland. In order to stimulate milk letdown, an intramuscular injection of 5 IU of oxytocin (Veyx Pharma, Germany) was given 10 min before milk collection. Rabbit milk composition (content of fat, protein, lactose, dry matter as solids non-fat, SNF) was investigated by infrared absorption instrument (Instrument MilkoScan FT 120 Foss Electric Denmark, according to the manufacturer’s manual). Determination of minerals

in ash after annealing was done by atomic absorption spectrometer (UNICAM 939 Solar STN 57 0532). Statistics Differences between the transgenic and nontransgenic groups were determined by ANOVA followed by Duncan´s multiple range test. Differences between groups at p<0.05 were considered as significant.

RESULTS and DISCUSSION To analyze milk composition from transgenic and non-transgenic rabbits, the content of fat, protein, lactose, SNF and minerals was determined (Table 1). There were no differences in the content of rabbit milk fat in the hPC transgenic rabbits (11.18 g/100 g) or hFVIII transgenic rabbits (11.48 g/100 g) compared to non-transgenic ones (10.40 g/100 g). Similarly, although the contents of rabbit milk protein (9.25 and 9.30 g/100 g respectively) and lactose (1.99 and 1.89 g/100 g respectively) were higher in the transgenic compared to non-transgenic (8.38 and 2.26 g/100 g respectivelly) rabbits, the differences were not significant. Also, there were no differences in mineral content (ash, Ca, P, Mg, Na) of milk between transgenic and non-transgenic animals (Table 2). We present a results about milk composition of transgenic (carrying human protein C or human factor VIII gene) rabbit females at first lactation. Specific transgenic over-expression of hPC or hFVIII in the mammary gland can be obtained without any strong influence on rabbit milk yield in different lactations, indicating that this technology can be applied to “pharmaceutical farming”

Table 1: Milk composition of transgenic and non-transgenic rabbits at the first lactation

Females

Fat

Protein

Lactose

SNF

g/100 g

11.18±0.24

9.25±0.25

1.99±0.33

12.08±0.24

9.30±0.25

1.89±0.03

12.04±0.24

8.38±0.15

2.26±0.03

12.09±0.23

Transgenic

(hPC) average

(n=8)

Transgenic

(hFVIII) average

(n=8)

Non-transgenic

average

(n=8)

11.48±0.24

10.40±0.22

Slovak J. Anim. Sci., 46, 2013 (1): 7-10

Original paper

(Dragin et al., 2005, Chrenek et al., 2007a). Rabbit milk composition varies depending on various factors, such as breed, nutrition, lactation stage or number of pups (Chrastinova et al., 1997; Lukafahr et al., 1983). To investigate difference in milk composition between transgenic and non-transgenic rabbit females, basic analysis of milk was performed at the same conditions. Our previous results on transgenic rabbit milk samples showed that all tested transgenic females produced either lower or higher concentrations of rhFVIII ranged from 5 to 1170 µg/ml, with variations among individual transgenic females (Chrenek et al., 2005, 2007b). Significant differences were found in the content of milk fat, protein and lactose. The higher variability in rhFVIII concentration may be explained by different copies of integrated gene, the site of transgene insertion or its genomic environment, what influence its expression (SalvoGarrido et al., 2004). Higher protein content in transgenic rabbit milk samples may be explained by the production of recombinant human factor VIII, although an expression of recombinant protein into the mammary gland of transgenic animals may not be automatically resulted in any increase in total milk protein content (Wilde et al., 1992). Significant differences obtained in our milk samples are in agreement with the physiological range of variability, where an average of fat content is 10-15 g/100 g, protein is 8-12.5 g/100 g, lactose 1.0-2.0 g/100 g and ash is 2-3 % (Davies, 1983). Palmer et al. (2003) recently reported that transgenic mice expressing recombinant human protein into mammary gland, under mouse WAP promoter,

exhibit defects in lactation and impaired mammary gland development. The WAP promoter was shown to be less efficient than the β-lactoglobulin promoter at driving the over-expression of recombinant human protein into milk (Barash et al., 1999). On the other hand, Van Cott et al. (2001) concluded that transgenesis and recombinant human protein secretion in milk was not connected with any abnormality concerning milk production, such as mastitis or other mammary gland disorders of transgenic pig. Mammary gland specific over-expression of IGF-I did not have also any impact on lactation performance in swine (Monaco et al., 2005). Our results showed that mammary gland specific transgenic over-expression of hPC or hFVIII can be obtained without any strong influence on rabbit milk yield and composition in several generations and at different lactations. Mammary gland specific over-expression of mWAP-hPC or mWAP-hFVIII gene constructs has only minor and transient effect on milk composition. These differences are in agreement with the physiological range of variability for rabbit milk composition.

ACKNOWLEDGEMENT This work was supported from the APVV0556-011 grant coordinated by the Slovak Research and Development Agency and international project A Collaborative European Network on Rabbit genome Biology (COST-RGB-NET).

Table 2: Milk mineral content of transgenic and non-transgenic rabbits at the first lactation

Females

Ash

g/100 g

2.12±0.02

0.33±0.01

0.31±0.01

0.04±0.02

0.11±0.01

0.38±0.04

0.33±0.01

0.04±0.02

0.12±0.02

0.41±0.01

0.31±0.04

0.04±0.02

0.11±0.03

Transgenic

(hPC) average

(n=8)

Transgenic

(hFVIII) average

(n=8)

Non-transgenic

average

(n=8)

2.02±0.02

1.88±0.02

Original paper REFERENCES BARASH, I. A. – FAERMAN, M. – RICHENSTEIN, R. – KAIRI, G. M. – DAMARY, M. – SHANI, M. J. – BISSELL. M. 1999. In vivo and in vitro expression of human serum albumin genomic sequences in mammary epithelial cells with β-lactoglobulin and whey acid protein promoters. Molec. Reprod. Dev., vol. 52, 1999, p. 241-252. CHRENEK, P. – VASICEK, D. – MAKAREVICH, A. – UHRIN, P. – PETROVICOVA, I. – LUBON, H. – BINDER, B. R. – BULLA, J. 2002. Integration and expression of the WAP-hPC gene in three generations of transgenic rabbits. Czech J. Anim. Sci., vol. 47, 2002, p. 45-49. CHRENEK, P. – VASICEK, D. – MAKAREVICH, A. V. – JURCIK, R. – SUVEGOVA, K. – BAUER, M. – PARKANYI, V. – RAFAY, J. – BATOROVA, A. – PALEYANDA, R. K. 2005. Increased transgene integration efficiency upon microinjection of DNA into both pronuclei of rabbit embryos. Transgenic Res., vol. 14, 2005, p. 417-428. CHRENEK P. – CHRASTINOVA L. – KIRCHNEROVA, K. – MAKAREVICH A. V. – FOLTYS, V. 2007a. The yield and composition of milk from transgenic rabbits. Asian-Australasian. J. Anim. Sci., vol. 20, 2007a, p. 482-485. CHRENEK, P. – DRAGIN, S. – KIRCHNEROVA, K. – CHRASTINOVA, L. – BOZIC, A. – FOLTYS, V. 2007b. Production of recombinant human factor VIII in the milk of transgenic rabbits. Slovak J. Anim. Sci., vol. 40, 2007b, p. 1-4. CHRASTINOVA, L. – SOMMER, A. – RAFAY, J. – SVETLANSKA, M. 1997. Avotan exploitation in rabbit nutrition. II. Nutrient digestibility and lactation performance of does rabbit. J. Farm Anim. Sci., vol. 30, 1997, p. 80-86. DAVIES, M. 1983. The composition of milk. Biochem. Lactation, T.B.Mepham. Elsevier, p. 71-117. DRAGIN, S. – CHRASTINOVA, L. – MAKAREVICH, A. – CHRENEK, P. 2005. Production of recombinant human protein C in the milk of transgenic rabbits from F3 generation. Folia Biologica (Krakow), vol. 53, 2005, p. 129-132. GARBER, K. 2000. RhFVIII deficit questioned. Nat. Biotechnol., vol. 18, 2000, p. 133-134.

Slovak J. Anim. Sci., 46, 2013 (1): 7-10 LUBON, H. – PALEYANDA, R. K. – VELANDER, W. H. – DROHAN, W. N. 1996. Blood proteins from transgenic animal bioreactors. Transfusion Medicine Reviews, vol. 10, 1996, p. 131-143. LUKEFAHR, S. – HOHENBOKEN, W. D. – CHEEKE, P. R. – PATTON, N. M. 1983. Characterization of straightbred and crossbred rabbits for milk production and associative traits. J. Anim. Sci., vol. 57, 1983, p. 1100-1107. MONACO, M. H. – GRONLUND, D. E. – BLECK, G. T.HURLEY, W. L. – WHEELER, M. B. – DONOVAN, S. M. 2005. Mammary specific transgenic overexpression of insulin-like growth factor-I (IGF-I) increase pig milk IGF-I and IGF binding proteins, with no effect on milk composition or yield. Transgenic Res., vol. 14, 2005, p.761-773. PALMER, C. A. – LUBON, H. – MC MANAMAN, L. 2003. Transgenic mice expressing recombinant human protein C exhibit defects in lactation and impaired mammary gland development. Transgenic Res., vol. 12, 2003, p. 283-292. SALVO-GARRIDO, H. – TRAVELLA, S. – BILHAM, L. J. – HARWOOD, W. A. – SNAPE, J. W. 2004. The distribution of transgene insertion sites in barley determined by physical and genetic mapping. Genetics, vol. 167, 2004, p. 1371-1379. TARANTO, S. – DI MEO, C. – STANCO, G. – PICCOLO, G. – GAZANEO, M. P. – NIZZA, A. 2003. Influence of age at weaning on caecal content characteristics and post-weaning performance and health of rabbits. Asian-Aust. J. Anim. Sci., vol.16 (10), 2003, p. 15401544. VAN COTT, E. K. – LUBON, H. – GWAZDAUSKAS, F. C. – KNIGHT, J. J. – DROHAN, W. N. – VELANDER. W. H. 2001. Recombinant human protein C expression in milk of transgenic pigs and effect on endogenous milk immunoglobulin and transferrin levels. Transgenic Res., vol. 10, 2001, p. 43-51. WILDE, C. J. – CLARK, A. J. – KERR, A. M. –KNIGHT, C. M. – MCCLENAGHAN, M. – SIMONS, J. P. 1992. Mammary development and milk secretion in transgenic mice expressing the sheep betalactoglobulin gene. Biochem. Journal, 1992, vol. 284, p. 717-720.

RABBIT AMNIOTIC FLUID AS A POTENTIAL ALTERNATIVE SOURCE OF BROADLY MULTIPOTENT STEM CELLS

J. Slamečka Jr.1,2, P. Chrenek1,2* Animal Production Research Centre Nitra, Slovak Republic Faculty of Biotechnology and Food Science, Slovak University of Agriculture, Slovak Republic

1 2

ABSTRACT A small animal model such as rabbit to serve as a biological model for in vivo tissue engineering experiments, would be desirable. The aim of our preliminary study was to describe a method of isolation of rabbit multipotent stem cells from the amniotic fluid and to examine the phenotype of these cells using flow cytometry. New Zealand White rabbit amniotic fluid was mixed with the culture medium and transferred into a T25 tissue culture flask. Analysis of rabbit stem cells was realized using specific antibodies and flow cytometry (BD FACSCanto II flow cytometry analyzer). Our preliminary resutls show rapid proliferation of amniotic fluid-derived adherent cells. We hypothesize that these cells have a broadly multipotent mesenchymal stem cell phenotype basing on their morphology and the expression of CD44. Furthermore, these cells can potentially serve as an excellent and amenable cell source for reprogramming experiments into naive induced pluripotent stem cells. Key words: rabbit; stem cell; amniotic fluid; proliferation

INTRODUCTION Amniotic ﬂuid is known to contain multiple cell types derived from the developing fetus (Priest et al., 1978; Polgar et al., 1989). Cells within this heterogeneous population can give rise to diverse differentiated cells including those of adipose, muscle, bone and neuronal lineages (DeCoppi et al., 2001; In’t Anker et al., 2003; Tsai et al., 2004; Prusa et al., 2004). De Coppi et al. (2007) described lines of broadly multipotent AFS cells, and used retroviral marking to verify that clonal human AFS cells can give rise to adipogenic, osteogenic, myogenic, endothelial, neurogenic and hepatic lineages, inclusive of all embryonic germ layers. Therefore, the authors postulated that they meet a commonly accepted criterion for pluripotent stem cells, without implying that they can generate every adult tissue. Amniotic fluid stem cells clearly display a unique

*Correspondence: E-mail: chrenekp@yahoo.com Peter Chrenek, Animal Production Research Centre Nitra, Hlohovecká 2, 951 41 Lužianky, Slovak Republic Tel.: +421 37 6546285 Fax: +421 37 6546189

phenotype that is mostly multipotent but borders on pluripotency. Due to this fact, it does not come as a surprise that they have been shown to be more amenable to reprogramming (Li et al., 2012) into the induced pluripotent stem cells that are virtually identical to embryonic stem cells (Takahashi and Yamanaka, 2006). Remarkably enough, Moschidou et al. (2012) showed that c-KIT+ human ﬁrst-trimester amniotic ﬂuid stem cells (AFSCs) can be fully reprogrammed to pluripotency without any ectopic factors at all, by culture on Matrigel in human embryonic stem cell (hESC) medium supplemented with only the small-molecule histone deacetylase inhibitor (HDACi) - valproic acid (VPA). The cells share 82 % transcriptome identity with hESCs and are capable of forming embryoid bodies (EBs) in vitro and teratomas in vivo. After long-term expansion, they maintain genetic stability, protein level expression of key pluripotency factors, high cell-division kinetics, telomerase activity, repression of X-inactivation, and

Received: January 23, 2013 Accepted: February 8, 2013

Original paper capacity to differentiate into lineages of the three germ layers, such as deﬁnitive endoderm, hepatocytes, bone, fat, cartilage, neurons, and oligodendrocytes. The authors conclude that AFSC can be utilized for cell banking of patient-speciﬁc pluripotent cells for potential applications in allogeneic cellular replacement therapies, pharmaceutical screening, and disease modeling. Amniotic fluid stem cells have also been demonstrated to represent a feasible cell source for advanced applications such as tissue engineering of living, autologous heart valves. The feasibility of using these cells for tissue engineering has been demonstrated in vitro as well as in vivo in sheep model (Schmidt et al., 2007; Schmidt et al., 2008; Weber et al., 2012). However, using sheep as a model for tissue engineering expreiments is logistically and financially cumbersome. Therefore, a small animal model such as rabbit to serve as a biological model for in vivo tissue engineering experiments, would be desirable (Slamečka and Chrenek, 2010). Here, we describe a method of isolation of rabbit multipotent stem cells from the amniotic fluid and we examine the phenotype of these cells using flow cytometry.

Material and Methods Cell Culture New Zealand White rabbit amniotic fluid was recovered from the uterus at about 23 day of gravidity using sterile syringe. The composition of the culture medium for the amniotic fluid stem cells was as follows: EBM-2 basal medium (CC-3156, Lonza) supplemented with 20 % fetal calf serum (FCS); recombinant growth factors: bFGF, EGF, R3-IGF-1; vitamin C (all are part of the medium kit); penicillin and streptomycin (15140122, Life Technologies). Rabbit amniotic fluid was mixed with the culture medium in proportion of 5:6, respectively. 5 ml of this mixture was then transferred into a T25 tissue culture flask. Approximately 5 days following plating, colonies of adherent cells start to be visible. At this point, a medium change is performed and from this point onwards, the medium is changed every day. On the day 10, the adherent outgrowths are after washing with PBS harvested using 2ml of Accutase (A1110501, Invitrogen) for 5 min at 37°C and 5 % CO2. The cells were counted and replated for the purpose of increasing the cell numbers. We found the optimum density of the cells to be 6-8×104/cm2 of cell culture surface. Cryopreservation Freezing of the rabbit amniotic fluid stem cells was performed using a medium consisted of 1:1 mixture of culture medium and freezing medium, respectively. The freezing medium consists of serum supplemented with

Slovak J. Anim. Sci., 46, 2013 (1):11-15 20 % DMSO (D2650, Sigma-Aldrich). Each cryovial contains about one million cells. Flow Cytometry For the purpose of flow cytometry analyses, the rabbit amniotic fluid stem cells are harvested using Accutase and washed with PBS. Then, the cells are fixed in 2 % paraformaldehyde (P6148, Sigma-Aldrich) for 20 minutes and washed in PBS. The cells were subsequently permeabilized in BD Perm Buffer III (558050, BD Biosciences) on ice for 30 minutes and then washed in PBS supplemented with 2 % FCS. The cells were then incubated with primary antibodies for 45 minutes on ice, washed twice and incubated with the secondary antibody for another 45 minutes on ice. Following washing, the cells were resuspended in PBS + 2 % FCS and analyzed using BD FACSCanto II flow cytometry analyzer. The antibodies used were the following: CD44 (sc-59758; SantaCruz Biotechnology), CD45 (304002; BioLegend), CD73 (344002; BioLegend), CD90 (328102; BioLegend), CD105 (sc-18838; SantaCruz Biotechnology), IgG1 kappa isotype control (400102; BioLegend), IgG2a isotype control (400202; BioLegend) and Cy2 secondary antibody (115-225-003, Jackson ImmunoResearch).

Results and Discussion The derivation of adherent rabbit amniotic fluid stem cells from cells suspended in the amniotic fluid was surprisingly rapid. While in case of humans, relatively small colonies of amniotic fluid stem cells just start to appear usually on the day 5 following the initial plating, the colonies of rabbit amniotic fluid stem cells, we observed on the day 5, were much bigger and comprised of considerably higher number of cells. We found two predominant types of morphology in the cultures of rabbit amniotic fluid stem cells – “spindleshaped” and “tile-shaped”. Spindle-shaped rAFSC grow in form of colonies comprised of the individual cells being more spread out with bigger gaps (Figure 1A). This morphology of the cells suggests a mesenchymal phenotype. The other morphology observed might indicate an epithelial phenotype (Figure 1B) but further experiments, preferably immunocytochemistry using labeled antibodies specific to epithelial cell markers, would have to be performed to confirm this hypothesis. Following passaging, the tile-shaped cells still appear (Figure 2B) but the proliferation rate of the spindle-shaped (Figure 2A) cells is higher and they will ultimately take over. Following 13 days of culture, a total of 6.85 million cells were derived and cryopreserved. We analyzed the expression of several markers typically used to profile mesenchymal stem cells – CD44, CD90, CD73, CD45 and CD105 (Figure 3). CD45

Slovak J. Anim. Sci., 46, 2013 (1): 11-15

Original paper

Fig. 1-2: Morphology of the cells recovered from rabbit amniotic fluid. 1A - Spindle-shaped rAFSC grow in form of colonies comprised of the individual cells being more spread out with bigger gaps. 1B The morphology observed might indicate an epithelial phenotype. The tile-shaped cells still appear (Figure 2B) but the proliferation rate of the spindle-shaped cells is higher and they ultimately take over

expression is typically not observed in mesenchymal stem cells as opposed to the cells of the hematopoietic lineage and therefore, it serves as a negative marker. We detected the expression of CD44 only out of all mesenchymal stem cell-reactive markers, the expression was 47.8 % (Figure 3B). We hypothesize that the other mesenchymal stem cell markers can still be expressed but the antihuman antibodies we used have not cross-reacted with the proteins. This assumption is based on the fact that the cells have been derived under conditions identical to those used to derive human amniotic stem cells and the fact that the morphology of the rabbit amniotic fluidderived adherent cells is â&#x20AC;&#x153;spindle-shapedâ&#x20AC;? typically associated with mesenchymal stem cells. Our preliminary results show rapid proliferation of amniotic fluid-derived adherent cells. We hypothesize that these cells have a broadly multipotent mesenchymal stem cell phenotype basing on their morphology, the

expression of CD44 (Figure 3B) and the fact that they have been derived under conditions that have been shown to suport the mesenchymal stem cell phenotype of the human amniotic fluid stem cells (Schmidt et al., 2007). However, this is not enough to confirm our hypothesis and therefore, we will seek for a more profound scrutiny of the phenotype of these cells. For that, a set of specific antibodies reactive against mesenchymal stem cell markers, will have to be designed. As rabbit-specific antibodies are scarcely available, an in-depth scrutiny of the cross-reactivity of anti-human or anti-mouse antibodies is going to have to be carried out which might prove to be a daunting challenge. Once such a set of antibodies is desinged, detailed multiplex flow cytometry analysis and immunocytochemistry confocal examination will be caried out. Rapidly proliferating broadly multipotent rabbit mesenchymal stem cells would prove to be valuable for tissue engineering and cell therapy

Original paper

Slovak J. Anim. Sci., 46, 2013 (1):11-15 experiments enabling rabbit to become a sought-after small-animal biological model for these applications. Furthermore, these cells can potentially serve as an excellent and amenable cell source for reprogramming experiments into naive induced pluripotent stem cells.

Acknowledgement This work was supported from the grants of Slovak Research and Development Agency: APVV LPP0119-09 and APVV-556-011.

References

Fig. 3: Expression of several markers typically used to profile mesenchymal stem cells – CD44, CD90, CD73, CD45 and CD105 on rabbit amniotic fluid-derived stem cells

DE COPPI, P. – BARTSCH, G. J. Jr. – SIDDIQUI, M. M. – XU, T. – SANTOS, C. C. – PERIN, L. – MOSTOSLAVSKY, G. – SERRE, A. C. – SNYDER, E. Y. – YOO, J. J. – FURTH, M. E. – SOKER, S. – ATALA, A. 2001. Human fetal stem cell isolation from amniotic ﬂuid. American Academy of Pediatrics National Conference, San Francisco, 2001, p. 210-211. DE COPPI, P. – BARTSCH, G. J. Jr. – SIDDIQUI, M. M. – XU, T. – SANTOS, C. C. – PERIN, L. – MOSTOSLAVSKY, G. – SERRE, A. C. – SNYDER, E. Y. – YOO, J. J. – FURTH, M. E. – SOKER, S. – ATALA, A. 2007. Isolation of amniotic stem cell lines with potential for therapy. Nature Biotechnology, 25, 2007, p. 101-106. IN ´T ANKER, P. S. – SCHERJON, S. A. – KLEIJBURGVAN DER KEUR, C. – NOORT, W. A. – CLAAS, F. H. – WILLEMZE, R. – FIBBE, W. E. – KANHAI, H. H. 2003. Amniotic ﬂuid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood, 102, 2003, p. 1548-1549. LI, Q. – FAN, Y. – SUN, X. – YU, Y. 2013. Generation of induced pluripotent stem cells from human amniotic fluid cells by reprogramming with two factors in feeder-free conditions. J. Reprod. Dev., vol. 59 (1), 2013, p. 72-77. MOSCHIDOU, D. – MUKHERJEE, S. – BLUNDELL, M. P. – DREWS, K. – JONES, G. N. – ABDULRAZZAK, H. – NOWAKOWSKA, B. – PHOOLCHUND, A. – LAY, K. – RAMASAMY, T. S. – CANANZI, M. – NETTERSHEIM, D. – SULLIVAN, M. – FROST, J. – MOORE, G. – VERMEESCH, J. R. – FISK, N. M. – THRASHER, A. J. – ATALA, A. – ADJAYE, J. – SCHORLE, H. - DE COPPI, P. – GUILLOT, P. V. 2012. Valproic acid confers functional pluripotency to human amniotic fluid stem cells in a transgene-free approach. Mol. Ther., 20 (10), 2012, p. 1953-67. POLGÁR, K. – ADÁNY, R. – ABEL, G. KAPPELMAYER, J. MUSZBEK, L. PAPP, Z. 1989. Characterization of

Slovak J. Anim. Sci., 46, 2013 (1): 11-15 rapidly adhering amniotic fluid cells by combined immunofluorescence and phagocytosis assays. Am. J. Hum. Genet., 45, 1989, p. 786-792. PRIEST, R. E. – MARIMUTHU, K. M. – PRIEST, J. H. 1978. Origin of cells in human amniotic fluid cultures: ultrastructural features. Lab. Invest., 39, 1978, p. 106-109. PRUSA, A. R. – MARTON, E. – ROSNER, M. – BETTELHEIM, D. – LUBEC, G. – POLLACK, A. – BERNASCHEK, G. – HENGSTSCHLÄGER, M. 2004. Neurogenic cells in human amniotic fluid. Am. J. Obstet. Gynecol., 191, 2004, p. 309-314. SCHMIDT, D. – ACHERMANN, J. – ODERMATT, B. – BREYMANN, C. – MOL, A. – GENONI, M. – ZUND, G. – HOERSTRUP, S. P. 2007. Prenatally fabricated autologous human living heart valves based on amniotic fluid derived progenitor cells as single cell source. Circulation, 116 (11 Suppl), 2007, p. 64-70. SCHMIDT, D. – ACHERMANN, J. – ODERMATT, B. – GENONI, M. – ZUND, G. – HOERSTRUP, S. P. 2008. Cryopreserved amniotic fluid-derived cells: a lifelong autologous fetal stem cell source for heart valve tissue engineering. J. Heart Valve Dis., 17 (4), 2008, p. 446-55.

Original paper SLAMEČKA, J. Jr. – CHRENEK, P. 2010. Embryonic stem cells – current knowledge and future prospects. Slovak J. Anim. Sci., 43, (2), 2010, p. 50-59. TAKAHASHI, K. – YAMANAKA, S. 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126 (4), 2006, p. 663-76. TSAI, M. S. – LEE, J. L. – CHANG, Y. J. – HWANG, S. M. 2004. Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic ﬂuid using a novel two-stage culture protocol. Hum. Reprod., 19, 2004, p. 1450-1456. WEBER, B. – EMMERT, M. Y. – BEHR, L. – SCHOENAUER, R. – BROKOPP, C. – DRÖGEMÜLLER, C. – MODREGGER, P. – STAMPANONI, M. – VATS, D. – RUDIN, M. – BÜRZLE, W. – FARINE, M. – MAZZA, E. – FRAUENFELDER, T. – ZANNETTINO, A.C. – ZÜND, G. – KRETSCHMAR, O. – FALK, V. – HOERSTRUP, S. P. 2012. Prenatally engineered autologous amniotic fluid stem cell-based heart valves in the fetal circulation. Biomaterials, 33 (16), 2012, p. 4031-4043.

The effects of electroejaculation on some physiological parameters (rectal temperature, respiratory and cardiac rates) in Ouled Djellal breed

S. BOUSSENA1, O. BOUAZIZ1, M. L. DEHIMI2, S. HIRECHE1, R. AIMEUR1, R. KABOUIA1 Laboratory of Animal Health and Production Management, Department of Veterinary Science, University Mentouri of Constantine, Algeria 2 Institute of Animal Husbandry Techniques (ITELV), Ain M‘lila, Algeria 1

ABSTRACT In Algeria, the need to improve reproductive performance in sheep breed Ouled Djellal requires better knowledge of their semen characteristics. Electroejaculation is a procedure used to collect semen from rams. The aim of this paper is to characterize the physiological response of these animals during conventional electroejaculation. To asses the effect of this technique on some physiological parameters (rectal temperature, respiratory and cardiac rates) in Ouled Djellal breeds, 10 rams were subjected to semen collection by electroejaculation. The technique was applied every two weeks, for 4 consecutive months. The studied physiological parameters were monitored at different stages: before, during and after electrical stimulation. The use of electro-ejaculation technique significantly induces changes in rectal temperature, heart rate and to a lesser degree respiratory rate (P<0.05), with different individual responses to electrical stimulation (P<0.05). Recent introduction of this method of semen collection in the Technical Institute of Animal Husbandry (ITELV Ain M‘lila, Algeria) makes our study important to evaluate its effects on animal comfort and sperm quality thereby making it easy to select the most adapted rams which gives the best semen quality, compared to other techniques for semen collection. Key words: Ouled Djellal; rams; semen; electroejaculation; physiological parameters; comfort

INTRODUCTION The study of reproductive performance in Ouled Djellal sheep breed requires a quantitative and qualitative evaluation of the male semen. Ram semen is collected mainly for two reasons; firstly for breeding soundness evaluation (fertility) and secondly to be used for artificial insemination (Matthews et al., 2003). Several techniques have been used for semen collection in mammals and birds, such as massage or manual stimulation of the accessory sex glands (Palmer et al., 2004), artificial vagina and electroejaculation (Terrill, 1940; Wulster-Radcliffe et al., 2001). The latter technique is now commonly used for the collection of semen from domesticated species (Austin et al., 1961; Healey and

Sadleir, 1966; Sundararaman et al., 2007; Damián and Ungerfeld, 2011) or wild animals (Wildt et al., 1984; Wildt et al., 1986). The electro-ejaculator has been used in an effort to facilitate semen collection (Hulet et al., 1964). Electroejaculation is described as painful by some authors (Mosure et al., 1998; Etson et al., 2004; Palmer et al., 2004; Palmer, 2005) and induces severe stress (Voisinet et al., 1997; Orihuela et al., 2009a). Stafford (1995) noted that the electroejaculation was not more aversive than part-shearing. Unfortunately, few studies have been devoted to assess the physiological response of animals after repeated stimulation. To assess the effect of this technique on rectal temperature, respiratory and cardiac rates in Ouled Djellal breed, we conducted a four months study on 10 rams.

*Correspondence: E-mail: boussena_sabrina@yahoo.fr Sabrina Boussena, Laboratory of Animal‘s Health and Production Management, Department of Veterinary Science, El Khroub (25100), University Mentouri of Constantine, Algeria Tel.: +213699798056 Fax: +21331961115

Received: August 7, 2012 Accepted: November 23, 2012

Slovak J. Anim. Sci., 46, 2013 (1): 16-21 Material and methods A total of 10 intact rams of an average age of 14 months and an average weight of 70.75 ± 7.31 kg (weighing between 57 and 80 kg), were used in this study. The animals belonged to the Institute of Animal Husbandry Techniques (Ain M‘lila, Algeria) and were kept in stall with an exercise area. These animals received a ration of 2 kg of good quality oat hay, and 800 g of concentrate per animal per day. Water was distributed twice a day. The rams had been previously vaccinated against enterotoxaemia and sheep pox, they were dewormed twice a year (against internal and external parasites). The collection of semen sample was performed once in every fifteen days with an electroejaculator of 9 volts working on battery (Medata Ram Ejaculator, Medata Systems Limited, England) with a dimension of 16.5 cm in length, 1.7 cm in diameter and 0.2 kg in weight. The device had a ring to the rectal tube to minimize any trauma. During semen collection temperature, heart and respiratory rates were recorded once in every fortnight using a thermometer and a stethoscope. During stimulation, the animal was kept lying on its right side, placed on two bales of hay, to facilitate the collection of semen sample and record heart and respiratory rates. Prior to practice of electroejaculation, animals were restrained, with a rope, by tightening the two front legs with the right hind one. The procedure was followed as given below: After being lubricated to minimize trauma, the rectal probe was inserted into the rectum forward and slightly downward with a rotation movement, to be in contact with the male accessory glands. After exteriorization of the penis from the sheath,

Original paper electrical pulses were applied that last for 2-5 seconds, alternating with rest periods of 5 seconds and so on, until obtaining suitable ejaculation and not seminal plasma only. Practically, rams ejaculate after the 3rd electrical stimulation. Rectal temperature of the animals was measured before, during and after each collection, using an electronic thermometer. During the procedure, cardiac rate was also determined on the left side in the heart’s projection area, using a stethoscope; this was achieved before, 20 minutes after the cessation and even during electrical stimulation. Respiratory rate is measured relative to the movements of the left flank that reveals the number of respiratory cycles (inspiration, expiration). This frequency was taken in the same manner as for the cardiac rate. The experiment was carried out according to the animal welfare regulations of the Department of Veterinary Science, University Mentouri of Constantine Algeria. Statistical analysis involved the calculation of descriptive statistics: mean, standard deviation, standard error, minimum and maximum. The calculation of correlation matrix and significance test was done using the Pearson correlation coefficient (Statistica, 1999; Graph Pad, 2007).

RESULTS and DISCUSSION Overall mean values of rectal temperature, respiratory and heart rates at different stages of stimulation are presented in table 1. Rectal temperature differed significantly (P<0.001) over the three stages: before, during and after stimulation (Table 1). We also found that it decreases during the stimulation and then increases slowly afterwards as shown in Figure 1.

Table 1: Effect of electro-ejaculation on rectal temperature, heart and respiratory rates (Mean ±SEM) at different stages of stimulation

Parameter (mean ±SEM)

Before stimulation

During stimulation

After stimulation

Temperature (°C)

39.57±0.075a

39.32±0.092b

39.45±0.094c

Respiratory rate

49.83±1.24a

46.45±1.01ab

53.5±1.62b

Heart rate

101.01±1.93a

98.11±2.98b

102.75±2.53c

a, b, c

Means within rows, not followed by the same superscript are significantly different

Original paper

Slovak J. Anim. Sci., 46, 2013 (1):16-21

Fig. 1: Monthly variations in rectal temperature

Fig. 3: Monthly variations in respiratory rate

Recording temperature allowed us to calculate the individual mean rectal temperature during the study period (Figure 2).

During the three stages, the respiratory rate was higher after stimulation in almost all subjects. The same finding was reported throughout the different months of the study (Figure 3 and 4).

40,5

Before stimulation During stimulation

60 39,5 50 Respiratory rate

Rectal temperature Â° C

After stimulation

38,5

40 Before stimulation

During stimulation

20 38 1st

2nd

3rd

4th

5th

6th

7th

8th

9th

10th

ram

Fig. 2: Individual variations in rectal temperature (mean Âą SEM)

Analysis of variance showed a significant difference (P<0.01, r = 0.81) between respiratory frequency before and after stimulation. So, we found a change of this parameter in rams when the animal was in lateral recumbency and did not undergo stimulation or stimulation was ceased (Table 1). In the four months study, respiratory rate differed depending on the stage of the stimulation. During that time, it seemed to be higher after stimulation and lower during its application. The respiration of animals through the seasons was always higher in the month of March as compared to winter season, during all stages of stimulation (before, during and after) (Figure 3).

After stimulation

10 0 1st

2nd

3rd

4th

5th

6th

7th

8th

9th

10th

ram

Fig. 4: Individual variations in respiratory rate (mean Âą SEM)

As for temperature, significant differences were found between heart rate before and during electrical stimulation (r = 0.75, P <0.05), before and after stimulation (r = 0.85, P <0.01) and during and after the stimulation (r = 0.77, P <0.05). The ANOVA test showed a significance decrease at the statistical level P < 0.05, suggesting that heart rate is strongly affected by electrical stimulation of 9 volts used to collect the semen. Throughout the study period, heart rate generally declined during the stimulation to reach its initial value before stimulation (Figure 5).

Slovak J. Anim. Sci., 46, 2013 (1): 16-21

Original paper

Fig. 5: Monthly variations in heart rate

The study of monthly variations in heart rate showed that during winter, the recovery of this parameter is obtained few minutes after cessation of stimulation (Figure 5). Individual differences (Figure 6) were observed in the three stages of the application of electroejaculation (P<0.05).

140 120

Heart rate

100 80 60

Before stimulation During stimulation

After stimulation

20 0 1st

2nd

3rd

4th

5th

6th

7th

8th

9th

10th

Ram

Fig. 6: Individual variations in heart rate (mean Âą SEM)

To our knowledge, few studies have been devoted to the influence of electroejaculation on rectal temperature in sheep. Lindsay (1969) reported that electroejaculation resulted in virtually no rise in rectal temperature. But our results are consistent with those described recently by Fumagalli et al. (2011) in deer. The explanation of these results appears to be related to the introduction of the electroejaculator probe which brought to room temperature. Knowing that the study period was characterized by low ambient

temperatures (December - March), thus, the rectal temperature was lowered only in situ, but in reality, it does not mean that the core temperature has changed. The temperature at the time of stimulation is generally lower than those recorded before or after the same stimulation. That appears to be due to the poor contact between the anal mucosa and the thermometer, following the introduction of the probe. At the individual level, rectal temperature presents significant differences prior to stimulation which continues to exist until the end of electrical stimulation, but it is still within the physiological norms. Thus, we can say that the temperature is not really affected by this technique. Contrary to Orihuela et al. (2009a) who found that respiratory frequency does not differ significantly (P>0.05) following stimulation. Significant changes before and after stimulation was recorded in our study. Monthly variations in respiratory rate are higher after stimulation and lower during its application. This seems logical; the frequency is reduced during electrical stimulation (Matthews, 1993) because the animal is retracted in order to reduce the pain caused by the stimulation, then once it stops, it tries to compensate by accelerating its respiration, not to mention the increase in respiratory rate due to fear. Significant differences existed between some of the rams (P<0.05). In accordance with our study, a significant change in heart rate during electroejaculation has been cited by Mosure et al. (1998) and Orihuela et al. (2009 a, b). This change associated with electroejaculation is due to the combination of muscle contraction and pain caused by stimulation (Mosure et al., 1998). Forced muscle contractions can also cause pain (Carter et al., 1990). Thus, we may conclude that heart rate can be used as a representative indicator of animal welfare and so did Manteca in his study (1998). Monthly variations in heart rate showed that after stimulation, this parameter is recovered very rapidly but only in winter. Palmer (2005) reported that it recovers 2 minutes after stopping stimulation. However, in March, it continues to rise even few minutes after cessation of stimulation. Some subjects showed small changes in their heart rate. This can be explained by individual differences in the same farm or even between farms, found by Rushen (1990), concerning the ability of animals to memorize or learn in response to a given treatment. So, the rams of Ouled Djellal breed do not have the same tolerance to this technique. It would be better to rely on this criterion, if we want to use this technique to collect semen in the insemination centres in Algeria. Some authors advocated the use of anaesthesia to reduce the resulting pain (Mosure et al., 1998; Falk et al., 2001; Etson et al., 2004; Orihuela et al., 2009b). At the end, it should be noted that due to lack of resources,

Original paper an important parameter could not be studied; it is the blood level of cortisol during electroejaculation. Several authors have identified a significant change in blood levels of cortisol during electroejaculation (Etson et al., 2004; Ortiz-de-Montellano et al., 2007; Orihuela et al., 2009a,b; Damián and Ungerfeld, 2011). On the contrary, Stafford (1995) and Stafford et al. (1996) proved in their experiments that the increase in plasma cortisol response to electroejaculation did not differ significantly from that caused by certain practices, such as mowing or restraining the animal in lateral recumbency.

CONCLUSION The results of this study showed that the technique of electroejaculation used to collect semen in Ouled Djellal sheep has led to changes of the studied physiological parameters, especially the rectal temperature, heart rate and at a lesser degree respiratory rate. However, with the progress of time, the rams seem to start to adapt to this technique. Individual variations exist. In addition, some animals show a marked tolerance to this technique. So, it would be interesting to select them for reproduction, if we wish to use this technique for semen collection in Ouled Djellal sheep in Algeria.

ACKNOWLEDGMENT This scientific work was supported by PNR project 1/U250/342. We gratefully acknowledge Dr. M.C. Abdeldjalil for his help in preparing this manuscript.

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Slovak J. Anim. Sci., 46, 2013 (1): 16-21 Use of oxytocin and cloprostenol to facilitate semen collection by electroejaculation or transrectal massage in bulls. Anim. Repro. Sci., vol. 80, 2004, p. 213-223. PALMER, C. W. 2005. Welfare aspects of theriogenology: Investigating alternatives to electroejaculation of bulls. Theriogenology, vol. 64, 2005, p. 469-479. RUSHEN, J. 1990. Use of aversion-learning techniques to measure distress in sheep. Applied Anim. Behav. Sci., vol. 28, 1990, p. 3-14. STAFFORD, K. J. 1995. Electroejaculation: A welfare issue. Surveillance, vol. 22, 1995, p. 15-17. STAFFORD, K. J. – SPOORENBERG, J. – WEST, D. M. – VERMUNT, J. J. – PETRIE, N. – LAWOKO, C. R. O. 1996. The effect of electroejaculation on aversive behaviour and plasma cortisol concentration in rams. N. Z. Vet. J., vol. 44, no. 3, 1996, p. 95-98. STATISTICA, 1999. Version 5.5, ed. Stat. Soft, France. SUNDARARAMAN, M. N. – KALATHARAN, J. – EDWIN, M. J. 2007. Attempts to achieve semen collections from incapacitated Boer bucks by electroejaculation. Asian J. Anim. Vet. Adv., vol. 2, no. 4, 2007, p. 244-246. TERRILL, C. E. 1940. Comparison of ram semen

Original paper collection obtained by three different methods for artificial Insemination. J. Anim. Sci., 1940, p. 201-207. VOISINET, B. – GRANDIN, T. – MORTIMER, R. – RUSK, C. 1997. Use of physiological measurement, behavioral observations and vocalizations to determine the aversiveness of electroejaculation in Angus bulls. J. Anim. Sci., 1997, vol. 75, Suppl. 1. WILDT, D. E. – MELTZER, D. – CHAKRABORTY, P. K. – BUSH, M. 1984. Adrenal-testicular-pituitary relationships in the cheetah subjected to anesthesia/ electroejaculation. Biol. Reprod., vol. 30, 1984, p. 665-672. WILDT, D. E. – HOWARD, J. G. – CHAKRABORTY, P. K. – BUSH, M. 1986. Reproductive physiology of the clouded leopard: II. A circannual analysis of adrenal-pituitary-testicular relationships during electroejaculation or after an adrenocorticotropin hormone challenge. Biol. Reprod., vol. 34, 1986, p. 949-959. WULSTER-RADCLIFFE, M. C. – WILLIAMS, M. A. – STELLFLUG, J. N. – LEWIS, G. S. 2001. Technical note: Artificial vagina vs a vaginal collection vial for collecting semen from rams. J. Anim. Sci., vol. 79, 2001, p. 2964-2967.

Slovak J. Anim. Sci., 46, 2013 (1): 22-30 ÂŠ 2013 CVĹ˝V ISSN 1337-9984

HERBAGE YIELD AND QUALITY OF Lablab Purpureus DURING THE LATE DRY SEASON IN WESTERN NIGERIA T. A. AMOLE1,2*, B. O. ODUGUWA1, O. SHITTU1, A. FAMAKINDE1, N. OKWELUM1, V. O. A. OJO2, P. A. DELE2, O. J. IDOWU2, B. OGUNLOLU2, A. O. ADEBIYI2 Institute of Food Security, Environmental Resources and Agricultural Research, University of Agriculture, Abeokuta, Nigeria 2 Department of Pasture and Range Management, University of Agriculture, Abeokuta, Nigeria 1

ABSTRACT The objective of this study was to determine the herbage yield and nutritional quality of Lablab purpureus during the peak of dry season in South Western Nigeria and to ascertain its capacity as dry season supplement for ruminant animals in that region. The experiment was carried out at the Livestock Research Farm, Federal University of Agriculture, Abeokuta from May 2008 till March 2009. The 3 treatments consisted of 3 cutting periods replicated four times. The average CP content of lablab varied from 180 g.kg-1 DM to 220 g.kg-1 DM with the leaf fraction in March having the highest CP content of 220 g.kg-1 DM as a result of sudden rainfall. The highest (2745 kg/ha) yield was recorded in January while the least dry matter content was recorded in February. The ash and the ether extract content of lablab during the late dry season in South West Nigeria ranged from 50 g.kg-1 DM to 90 g.kg-1 DM and 55 to 80 g.kg-1 DM, respectively. The NDF, ADF and lignin content increased significantly (P<0.05) with advancing the harvesting stage with stem fraction higher (P<0.05) than that leaf fraction. The herbage yield and quality of L. purpureus in terms of its crude protein, fibre and minerals from this study shows that the appropriate time to harvest and use lablab as supplement to animal diet during the late dry season is January, which has the highest herbage yield and quality. Key words: forage legume; Lablab purpureus; dry season; herbage yield; quality

INTRODUCTION Poor nutrition is a major constraint to livestock production in small-holder crop-livestock farming system, especially during the dry season when available feed quantity is low and quality extremely poor (Alhassan, 1987). Basically this is due to the dependence of livestock on naturally available feed resources and little development on forage crops for feeding to animals (Whiteman, 1980). Normally it is during the dry season when problems such as sickness and weight loss due to poor dietary profile, scarcity of forages, reduction in yield and quality of forages arises. The productivity of Nigerian livestock is below its genetic potential, principally due to poor nutrition and

inadequacy of good quality feed (Lamidi et al., 2005). The feed shortage is most pronounced in the rural areas where the arable land is used for growing food and cash crops and not for fodder production (Kibiria et al., 1994). One of the ways of improving the utilization of such crop residue is by proper supplementation with leguminous forage (Poppi and McLennan, 1995). High quality sown forage such as leguminous fodder has been found to provide adequate dry season supplementation and improve the productivity of grazing cattle. The legume can be sown as pure stand in protein banks or undersown in food crops. It can be grazed, harvested and fed fresh or stored as hay or silage (Harricharan et al., 1988). As a consequence of

*Correspondence: E-mail: gokeamole@yahoo.com T. A. Amole, Institute of Food Security, Environmental Resources and Agricultural Research, University of Agriculture, Abeokuta, Nigeria

Received: September 4, 2012 Accepted: November 5, 2012

Slovak J. Anim. Sci., 46, 2013 (1): 22-30

Original paper

different biochemical pathway of carbon fixation during photosynthesis, nitrogen fixing legume have higher concentration of cellular protein than tropical grasses (Bjorkman et al., 1976 ), as such tropical legume are rich in protein which is limiting nutrient in tropical animal diet most especially during the dry season. They are more digestible and enhance dry matter intake by grazing animal. Lablab purpureus combines a great number of qualities that can be used successfully under various conditions. Its first advantage is its adaptability, not only it is drought resistant, it is able to grow in diverse range of environmental conditions worldwide. Staying green during the dry season, it has been known to provide up to six tones of dry matter per hectare (Murphy and Colucci, 1999). Being palatable to livestock, it is an adequate source of much needed protein and can be utilized in several different ways. In several experiments, it has been observed to increase livestock weight and milk production during the dry season (Murphy et al., 1999). Lablab is a legume that thrives well in the dry season between November and February in the northern Nigeria. It is drought resistant and is usually sown after the normal cropping season, thereby acting as a buffer crop for ruminant feed during the dry season (Adu et al., 1992). However its productivity during the dry season has not been evaluated in the western part of Nigeria which is known to have high rainfall and humidity with consequently short period of dry season. It is then necessary to determine the effects of seasonal change on the herbage yield and the nutritive value of Lablab purpureus.

Material and Methods

Fig. 1: Agrometrological observation at Ogun-Osun River Basin Development (OORBD)* in 2008 R/F: Rainfall (mm); MT: Mean Temperature (0°C); R/H: Relative humidity (%) Source: Ogun-OsunRiver Basin Development, Alabata Road, OgunState * 5 km from experimental site

Fig. 2: Agrometrological observation at Ogun-Osun River Basin Development (OORBD)* in 2009 R/F: Rainfall (mm); MT: Mean Temperature (0°C); R/H: Relative humidity (%) Source:Ogun-OsunRiver Basin Development, Alabata Road, OgunState * 5 km from experimental site

The experiment was carried out at the Teaching and Research Farm, University of Agriculture, Abeokuta (UNAAB), located on latitude 7°13’49.46”N, longitude 3°26’11.98”E of Ogun State, Nigeria (Google Earth, 2006). The research site was located in the derived savannah zone of Southwest Nigeria with monthly rainfall which ranged from 120 mm in May to195 mm in September and mean monthly temperature ranging from 22.5° to 33.7°C. The area is characterized by bimodal rainfall pattern which peaks in July and September and a major dry season between November and March. The relative humidity in the rainy (late March-October) and dry (November-early March) season ranged between 63-96 % and 55-84 %, respectively. The rainfall data for the two years of experiments (2008 and 2009) are presented in Figures 1 and 2. Experimental Plot The site for planting was cleared and ploughed twice. Thereafter, the land was harrowed and levelled. A total of 12 plots, each measuring 20 m x 25 m was measured out and demarcated by 2 m spaces between plots and 3 m spaces between blocks. Three core samples of soil (0 – 15 cm) were randomly collected from the plots before planting. These were bulked for each block and analyzed for physical (particle size) and chemical properties (pH, total N, organic carbon, C: N ratio, available P, available N, cation exchange capacity and acidity).

Original paper

Slovak J. Anim. Sci., 46, 2013 (1):22-30

The seed of Lablab purpureus var. Highworth, which has a purple band near the leaf axil, purple flowers and black seeds, was used in this experiment. It is an early flowering line with high seed-yielding ability; it is suitable for pulse production and forage uses. The seeds were obtained from National Animal Production Research Institute (NAPRI), Zaria and were planted at the rate of 15 kg/ha and spacing of 50 x 50 cm with one seed per hole. Twelve (12) experimental plots each measuring 10 x 20 m was established in May 2008 with 3 treatments which were 3 cutting periods replicated four times. Single superphosphate (SSP) fertilizer was applied in July 2008. The site was weeded and fenced to prevent accidental grazing. The quality and quantity evaluation study was conducted in the first 3 months of the following year (during the dry season period of January to March in South Western Nigeria). Data Collection Evidences of flowers and green pods were noticed in November and December (6-7 months after planting). Since the focus of this study was not after grain production, flowers and pods were separated at each harvest and were not included in the yield estimate. Herbage yield was estimated by cutting from 1 m2 quadrants randomly thrown five (5) times on each plot using knife, with each stand cut at 5 cm above the ground level every month. Samples were separated into leaf and stem from each replicate and weighed in the laboratory using the sensitive scale, to determine the leaf to stem ratio. Two hundred grams (200 g) sub-sample was taken from each replicated leaf sample and 100 g from each replicated stem sample. The sub-samples was put in individual paper bag and dried in the drying cabinet till constant weight was attained. The dry matter yield of each replicate was calculated as:

Oven dried sub-sample Fresh sub - sample

100

The samples were oven dried, hammer-milled and sieved through a 1 mm mesh and were used for the analysis. Dry matter (DM) content was determined by drying at 80°C for 48 h (AOAC, 1995) while ash content was determined with a muffle furnace at 510°C for 18 h. Crude protein (N % x 6.25) content in the samples was determined by LECO FP-200 Analyzer (St Joseph, MI, USA) while oil (as ether extract) was extracted with petroleum spirit (b.p. 40 to 60°C) by the Soxhlet method (AOAC, 1995). The method of Van Soest and Robertson (1991) was used to determine the neutral detergent fibre (NDF), the acid detergent fibre (ADF) and acid detergent lignin (ADL). Cellulose was taken as the difference between ADF and ADL while hemicellulose

was calculated as the difference between NDF and ADF. The samples were thoroughly washed in water to remove extraneous matter and then dried at 60°C for 2-3 days in an oven before milling. The samples were digested by nitric and perchloric acids mixture (ratio = 4.1 v/v) and the concentrations of the minerals (Calcium (Ca), Potassium (K), Phosphorus (P) and Magnesium (Mg) in the samples were determined by an Atomic Absorption Spectrophotometer (Buck scientific model 200a; Buck Scientific, East Norwalk, CT 06855, USA). The data obtained was analyzed using Two Way Analysis of Variance (ANOVA) in a completely Randomized Block Design. Analysis of variance of growth and nutritional parameters was performed using the general linear models procedure of SAS (1996). Yij = a + ßi + tj + eij Where: Yij = dry matter yield and nutritional parameters a = general mean of the treatments ß = block effects i = I, II, III, IV (blocks) t = treatment effects (days post germination) j = 1,2,3, (sampling period) e = experimental error Differences between means were compared using the Duncan’s Multiple Range Test (Duncan 1995).

Results and discussion The average crude protein (CP) content of lablab varied from January to March ranging from 180 g.kg-1 DM to 230 g.kg-1 DM with the leaf fraction in March having the highest CP content of 226 g.kg-1 DM (Table 1). The CP content in the leaf fraction was higher than that of the stem throughout the experiment. The CP content of the leaf fraction decreased (P<0.05) from January to February and then increased (P<0.05) later in March, while there was significant (P<0.05) reduction in the CP content of stem fraction as lablab increased in age during experimental period. The dry matter yield (DMY) of lablab both leaf and stem reduced from January (1028 kg/ha and 2745 kg/ha respectively) to February and then increased in March. The highest (P<0.05) yield was recorded in January. The dry matter content of lablab (leaf and stem fraction) in January was comparable (P>0.05) with that of March while the least dry matter content was recorded in February. The crude fibre content of Lablab purpureus during the dry season ranged from 292 g.kg-1 DM to 371 g.kg-1 DM in the leaf fraction while the stem fraction

Slovak J. Anim. Sci., 46, 2013 (1): 22-30

Original paper

Table 1: The herbage dry matter yield and approximate composition (g.kg-1 DM) of L. purpureus during the late dry season Treatment DMY (kg/ha) DMC CP CF ASH EE NFE

GE (kJ kg-1 DM)

JAN

Leaf

1028c

775b

215b

292d

55.0f

55.6e

183a

47.3d

Stem

2745a

777b

204c

304cd

80.9c

529f

159b

53.2c

FEB

Leaf

862c

720c

204c

310c

65.6e

70.4b

150b

66.6b

Stem

1743

737

195

365

83.5

60.6

960

66.6b

MAR

Leaf

977c

755b

226a

371b

68.7d

75.6a

589d

67.8b

Stem

2123b

771b

181e

422a

86.3a

70.4c

473d

76.2a

SEM

0.57

0.01

0.05

0.00

0.01

0.05

0.02

a, b, c, d, e, f

means with different superscripts in the same column differ significantly (P<0.05) CP: Crude Protein; CF: Crude Fibre ASH: Ash content; EE: Ether Extract ; NFE: Nitrogen Free Extract ; DMY: Dry Matter Yield; DMC: Dry Matter Content; GE: Gross Energy; JAN: January; FEB: February; MAR: March

ranged from 304 g.kg-1 DM to 422 g.kg-1 DM. The CF content of the stem and leaf fraction increased (P<0.05) monthly. The ash and the ether extract content of lablab during the late dry season in South West Nigeria ranged from 55 g.kg-1 DM to 90 g.kg-1 DM and 55.6 g.kg-1 DM to 80 g.kg-1 DM, respectively. The ash content of the stem was higher (P<0.05) than the leaf fraction throughout the experiment. However, the ether extract content of the leaf was higher (P<0.05) than the stem fraction throughout the dry season period. The gross energy of the leaf content increased (P<0.05) from 47.3 kJ.kg DM in January to 68.2 kJ.kg DM in

March. The stem fraction followed similar trend and reached the highest (P<0.05) value of 76.2 kJ.kg DM in March. The NDF content of lablab during the late dry season increased significantly (P<0.05) from 660 g.kg-1 DM in leaf fraction harvested in January to 700 g.kg-1 DM % in stem fraction harvested in March. ADF and lignin followed similar trend with its contents in the stem fraction higher (P<0.05) than that of leaf fraction. The hemicellulose content reduced (P<0.05) both in the leaf and the stem fraction as lablab increased in age from January to February. The cellulose content increased in both leaf and stem fraction and reached its peak (310 g.kg-1 DM) in March.

Table 2: The fibre composition (g.kg-1 DM) of L. purpureus during the late dry season

Treatment

NDF

ADF

LIGNIN

HEM

CEL

JAN Leaf

661

295

41.4

366

254d

656f

358c

51.2c

298d

307bc

FEB Leaf

686d

354d

48.4e

333b

305c

Stem

695

361

53.1

334

308b

MAR Leaf

691c

363b

49.7d

328c

313a

701a

369a

55.2a

332b

314a

0.01

0.02

0.01

Stem

SEM

a, b, c, d, e, f means with different superscript in the same column differ significantly (P<0.05) NDF: Neutral Detergent Fibre; ADF: Acid Detergent Fibre; HEM: Hemicellulose; CEL: Cellulose

Original paper

Slovak J. Anim. Sci., 46, 2013 (1):22-30

Table 3: The mineral composition of L. purpureus (g.kg-1 DM) during the late dry season

Treatment

JAN

Leaf

5.10e

2.40c

3.10d

2.00d

Stem

7.1d

1.60b

2.90c

3.40c

Leaf

9.4bc

2.30b

2.50ab

3.40c

Stem

8.7

1.50

2.80

4.10b

Leaf

10.1ab

2.30b

2.60a

4.50b

Stem

10.7a

1.60a

3.00a

6.20a

0.01

FEB MAR

SEM

means with different superscript in the same column differ significantly(P<0.05) Ca: Calcium; K: Potassium; P: Phosphorus; Mg: Magnesium

a, b, c, d , e

The mean Ca concentration in both the leaf and stem fraction increased as the dry season progressed. However, the two fractions became comparable (P>0.05) from February to March with 10.1 g.kg-1 DM and 10.7 g.kg-1 DM for leaf and stem, respectively. Concentration of K of the leaf fraction was higher (P<0.05) than the stem fraction in each month of the experiment. However, the K concentrations of leaf and stem were similar in February and March. Concentration of Mg in the leaf fraction ranged from 2.0 to 4.5 g.kg-1 DM which were significantly (P<0.05) lower than values recorded in the stem fraction in the dry season. As the dry season progressed, concentration of Mg increased (P<0.05) in each fraction. Phosphorus (P) concentration ranged from 2.6 to 3.1 g.kg-1 DM in the leaf fraction with significant reduction from January to March, while the stem fraction fluctuated (P<0.05) in the dry season. The dry matter yield of lablab observed in the late dry season ranged from 1028 and 2745 kg DM/ha. These values were similar to the values reported by Karachi (1983) who reported that total green DM yields ranged from 2000 to 12000 kg ha-1, and most of the yield was stem with the green leaf DM yields ranging from 400 to 3300 kg ha-1. The biomass yield of Lablab purpureus (cv. Rongai white) in this study was found to be lower than 4700 kg/ha 17 weeks after sowing recorded during dry season in Honduras (Murphy, 1998) while Nworgu and Ajayi (2005) also reported 48.66 and 44.58 t/ha/yr for Lablab purpureus at Ibadan in 2001 and 2002, respectively. Amodu et al., (2005) reported a yield of 4.5 to 4.9 t/ha in November at a location in the Northern part of Nigeria. However, Kiflewahid and Mosimanyana (1987) reported an average dry-matter yield (ton/ha) of 1.08 to 1.94 from 0, 100 and 250 kg/ha rate of SSP fertilizer during low seasonal rainfall and distribution

patterns in the project areas (262 to 414 mm rainfall) of Botswana, which are in agreement with the results of this study. Variations in the yields could be attributed to the level of soil fertility, climatic zones, seasons and agronomic practices adopted. Cameron (1988) and Mayer et al. (1986) noted that dry matter yield per hectare varies with rainfall, soil condition and time of seeding. Differences in the yield could also be as a result of much shedding of leaves during the peak of the dry season as observed in the present study. Notwithstanding, the dry matter yield recorded in the present experiment is substantial as supplements for grazing animals in dry season depending on the breed and the number of herds. Such DM yield of L. purpureus at the peak of dry season can be attributed to its aggressive and vigorous growth habit (Skerman et al., 1988) and its ability to maximize low soil moisture content for growth as drought resistant. Nworgu and Ajayi (2005) reported mean monthly rainfall of 125 mm to 0.65 mm for August to December 2001 and 2002 at Ibadan (40 km from where this experiment was carried out) and concluded that such amount was adequate for the growth of a drought resistant forage legume. A significant difference was observed between crude fibre content of leaf and stem throughout the experimental period. The fibre composition of L. purpureus during the late dry season in western Nigeria was higher than that reported by Nworgu and Ajayi (2005), and that of Murphy and Colucci (1999) who summarized the crude fibre of the whole lablab plant as 27.8 %, which could be attributed to the time of harvest. Crude fibre content of legumes generally increases with maturity (Minson, 1990). High temperatures decrease the soluble carbohydrate content of plants, resulting in increased fibre content and decreased digestibility. The fibre levels

Slovak J. Anim. Sci., 46, 2013 (1): 22-30 recorded in leaves are lower than that in the stem fraction. The quality of stems is largely affected because of their structural function while the leaves are metabolic organs (Shehu et al., 2001). Therefore, selection for immature leaves with low cell wall content by grazing animals will improve nutritive value and digestibility over that indicated by a whole plant crude fibre value. The NDF and ADF values recorded in this study were comparable to 51.2 % and 69.2 % reported by Karachi (1997) for lablab harvested 100 days after planting during the short and long rain in Kenya. These values were higher than the average values of 43 % and 38.6 % for the lablab plant and various fractions on dry matter basis reported as Murphy and Colucci (1999). The increase in NDF content of the stem from January to March is related to physiological changes that occur as plant ages, that lead to a decrease in cell cytoplasm highly soluble components (cell contents), accompanied by an increase in cell wall fibre components (Nogueira et al., 2000). The cell wall of the leaf fraction exhibits higher proportions of hemicellulose (NDF ADF) and lower proportions of cellulose (ADF - ADL) than the stem fractions. These differences are reflected in higher ratios of hemicellulose to cellulose in the leaf than in the stem. Karachi (1997) also reported a greater neutral detergent fibre (NDF) in stem than the leaf, due to the higher amounts of fibre and lignin in it. Hall et al. (1997) also reported that soluble fibre decreased with maturity in stems but not in leaves. The CP content of lablab in the late dry season of South Western Nigeria ranged between 18 % and 22 % and these values fall within the range reported in the literature for tropical herbaceous legumes (Topps and Oliver, 1993; Norton and Poppi, 1995). The CP content of lablab in this study agrees with results obtained by Hendricksen and Minson (1985) and Cameron (1988) who reported leaf protein in lablab to be 21.6 % - 27.9 % and 15 % - 33 %, respectively. The average stem crude protein content falls within the range as reported by Murphy and Colucci (1999). The observed decline in CP content of lablab with increasing maturity is in agreement with results from another study (Khorasani et al., 1997). This decline is attributed to an increase in cell wall accumulation while cell contents decline (Buxton, 1989). Zinash et al. (1995) also reported the decline in CP content of the pasture along with increasing age of harvesting, which might be due to the dilution of the CP content by increasing structural carbohydrates of forages harvested at late maturity (Hassan et al., 1990). The results of this study showed that the CP content of leaf was higher than that of stem. This was in agreement with the report of Van Soest (1994) that the leaf fraction of legumes often has a better nutritional quality in comparison to the more fibrous stems. The leaf fraction of legumes has been reported

Original paper in earlier studies to have a higher CP content than the stems (Adjei and Fianu, 1985; Cameroon, 1988). The crude protein content of the leaf and stem of lablab decreased as the dry season advanced from January to February. It has been established that as legumes mature, the content of protein decreases (Milford and Minson, 1968). However, the increase in the CP content of the leaf fraction in March (22.60) could be as a result of sudden rainfall (92 mm) experienced in early March in 2009 (Figure 1) which brings about the emergence of new plant materials. Thus it appears that the rain had an effect on the protein profile of the leaf fraction through the emergence of new plant material. This implies that lablab could make use of the minimum available precipitation for new plant growth. The protein content of lablab during the late dry season as expressed in this experiment is in excess of that proposed as the minimum requirements for lactation (120 g CP/kg DM) and growth (113 g CP/kg DM) in ruminants (ARC, 1984). This makes it good source of protein when given to ruminants as protein supplements to low quality roughage thereby reducing farmersâ&#x20AC;&#x2122; cost in procuring of concentrate in dry season. Phosphorus (P) and potassium (K) content of lablab was considerably lower than the value reported by Nworgu and Ajayi (2005) who reported that L. purpureus contain 0.33 % to 0.41 % P and 0.24 % to 0.28 % K in the early dry season. The increase and decrease in some mineral composition could be attributed to the age and time of maturity of the plant. The magnesium content of the leaf was lower than that of the stem throughout the experiment. However, mean Mg concentration in both fractions of lablab sampled in the dry season were higher than the suggested critical level of 0.2 % (McDowell and Arthington, 2005) for ruminants in the tropics signifying that lablab could adequately supply required amount of Mg for ruminant animals during the dry season which is characterized by low quantity and quality forage in turn affecting the livestock industry in terms of weight gain, reproduction, growth rate, productivity. Mean K concentration in lablab was lower than the critical level established (McDowell and Arthington, 2005), indicating that the forage legume is low in K concentration. However, McDowell and Valle (2000) reported that there are very few confirmed reports of K deficiency for ruminants grazing exclusively on forages. In beef cattle, a severe deficiency of K is unlikely but a marginal potassium deficiency results in decreased feed intake and retarded weight gain (NRC, 1996). With maturity, mineral concentration declines due to a natural dilution process and the translocation of minerals to the root system (Pastrana et al., 1991). Forages are generally good sources of calcium, and legumes are higher in Ca content than grasses.

Original paper The calcium content in forages is affected by species, portion of plant consumed, maturity, quantity of exchangeable Ca in the soil, and climate (Minson, 1990). The concentration of Ca reported in this trial is similar to the result of Nworgu and Ajayi (2005). Calcium concentration recorded in this study increased as the dry season progressed while Temesgen and Mohammed (2012) observed seasonal differences for Ca with concentration in the wet season being higher than that of the dry season. Based on the lower limits of ruminant requirements (McDowell and Arthington, 2005), both leaf and stem fractions contained higher concentration of Ca needed to meet ruminant requirements. Nworgu and Ajayi (2005) reported that Lablab purpureus can withstand the dry season better than Centrosema pubescens, Centrosema pascuorum and Aeschynomene histrix with higher Ca concentration than some tropical forage legumes. The ash content of the stem was higher than the leaf fraction throughout the experiment. The decline in ash content with increasing maturity could be due to a natural dilution process as dry matter accumulation outstrips mineral uptake as the forages mature (Bittman et al., 1988). Alia et al. (2007) also reported that the ash content of the plant parts browsed by camels during the wet season was higher compared to those browsed during the dry season.

Conclusion The potential of any feed to support animal production depend on the quality consumed by the animal and extend to which the feed meets energy, protein, minerals and vitamin requirement (Minson, 1990). Nutritive quality range varies from area to area, between seasons and growing stages. In many cases determination of crude protein, fibre fractions and minerals is sufficient to give an adequate assessment of forage quality (Sileshi et al., 1996). The herbage yield and quality of L. purpureus in terms of its crude protein, fibre and minerals from this study shows that the appropriate time to harvest and use lablab as supplement to animal diet during the late dry season is January which has the highest herbage yield and quality. Lablab could as well be fed as supplement throughout the dry season due to its persistence and quality. A sustainable way of improving the feeding value of poor quality crop residues and pastures as feed for livestock is through supplementation with forage legumes in which L. purpureus has been tried, proved and now recommended for livestock feeding during the late dry season.

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SOMATIC CELL COUNTS IN MILK OF DAIRY COWS UNDER PRACTICAL CONDITIONS

V. Tančin1,2* Animal Production Research Centre Nitra, Slovak Republic Slovak University of Agriculture in Nitra, Slovak Republic

1 2

ABSTRACT Knowledge related to udder health in dairy practice is considerably limited. The aim of the experiment was to study the changes of somatic cell counts (SCC) in milk in relation to the month of year, stage of lactation and parity under the practical conditions. Also the frequency of incidence of high SCC during the year was observed. The effectiveness of dry therapy and calving process on SCC was evaluated as well. The experiment lasted from January 2011 till January 2012 involving 146 dairy cows. From each milked cow two milk samples per month were collected to measure SCC during experimental observation. In total 2306 samples were collected during the experimental period. The SCC significantly increased with advanced numbers of lactation. The months of the year (season) significantly influenced the SCC in dairy cows. There was a significant increase of SCC during the period from May till July as compared with winter months of the year. The lowest value of SCC was measured at second month and the highest at tenth month of lactation. Also the first month of lactation was characterized by higher SCC as compared with second one. In conclusion, the high percentage of cows had SCC over 4.105ml-1 and considerable effect of season indicate insufficient preventive mastitis control programme in dairy farm and therefore the high risk for milk quality on farm. Key words: cow; somatic cells; mastitis; season; lactation

INTRODUCTION Milk and milk products intake by consumers is crucial for economic profit of producers. Therefore the positive development of milk intake by consumers should be stimulated by the high quality of raw milk in dairy practice. There are many factors influencing milk and milk products consumption (Kubicová and Dobák, 2012). Somatic cells are considered to be the cells of udder tissue (epithelial cells) and also cells coming from the blood like neutrophil, macrophages, lymphocytes. The white cells of udder are the most important part of udder immunity against pathogens penetrating through teat canal to the udder cistern (Kelly and McSweeney, 2002; Green et al., 2004). Therefore the increased somatic cell count (SCC) represents the response of

*Correspondence: E-mail: tancin@cvzv.sk Vladimír Tančin, Animal Production Research Centre Nitra, Hlohovecká 2, 951 41 Lužianky, Slovak Republic Tel.: +421 903 546 401

udder tissue to the presence of pathogens causing the inflammation of udder called mastitis (Pyörärlä, 2003). Mastitis represented by increased SCC significantly reduces milk synthesis and therefore milk production (Tančin et al., 2007) and milk composition especially content of lactose, chlorides (Barkema et al., 1999) and technological quality of milk (Santos et al., 2003). Curd yield was found to be significantly lower in the infected glands than in the uninfected ones (Leitner et al., 2004). The SCC could be considered as a main trait of udder health and milk quality (O´Brien et al., 2001). In general it is accepted that SCC is a gold standard at diagnostics of any form of mastitis in udder (Pyörärlä, 2003). On the other hand the SCC in milk are influenced by other internal and external parameters like stage of lactation, parity, teat position, milk flow kinetics (Tančin et al., 2007a,b) and frequency of milking (Hogeveen

Received: November 29, 2012 Accepted: February 8, 2013

Original paper et al., 2001), but these factors do not increase SCC dramatically as the mastitis do. The SCC in dairy practice is mostly related to the price of milk received from dairy and therefore if SCC is below the limit (4.105 cells.ml-1) the farmers are not dealing with SCC seriously. Therefore data from dairy practice could be useful for scientific evaluation to see more critical points that could be involved in mastitis prevention programmes. The aim of the experiment was to study the changes of SCC in milk in relation to the month of year, stage of lactation and parity under the practical conditions. Also the frequency of incidence of high SCC during the year was observed. The effectiveness of dry therapy and calving process on SCC was evaluated as well.

MATERIAL AND METHODS The experiment was realized in a dairy farm in practice. The experiment included Red Holstein cows on the farm with average annual milk production of 7200 kg. The experiment lasted from January 2011 till January 2012 on 146 dairy cows in their first lactation (35 %), second lactation (21 %), third lactation (19 %) and fourth and more lactations (25 %). The dairy cows were housed in free-stall housing system. Animals were milked two times a day in 2 x 4 autotandem milking parlour. The parlour was equipped with automatic devices for machine pre-stimulation and automatic cluster removal. The milking routine consisted from udder washing with water from hose, cleaning with towel and fore-stripping. From each milked cow two samples of milk per month were collected to measure somatic cell counts during experimental observation. In total 2306 samples were collected during the experimental period and were analyzed on Fossomatic 5000. Animals on the basis of SCC were divided into four groups: low (SCC < 2.105 cells.ml-1), middle (SCC between 2.105 and 4.105 cells.ml-1), high (SCC between 4.105 and 106 cells.ml-1) and highest (SCC > 106 cells.ml-1) to describe the frequency of possible udder health problems during experimental period. The effectiveness of dry therapy and calving process was studied through frequency of distribution of cows with different SCC in above mentioned groups. Cows were evaluated on the basis of three months average values of SCC before drying and after calving. Also the groups of animals according to the parity numbers were classified as primiparous cows and cows on their second and third lactation. Last group represented cows on their forth and more lactations. Stage of lactation was classified as first ten months of lactation and thirteen months were included to evaluate the effect of the year.

Slovak J. Anim. Sci., 46, 2013 (1):31-34 Obtained data were processed by Microsoft Excel program and statistically evaluated by SAS/ 8.2 (2002). The model was tested by using Fisher’s F-test. Differences between the levels of the effects were tested by Scheffe multiple range test for studied traits. Data are presented as least square means ± standard error. For evaluation of somatic cells the following model was used: y= Xβ + Zu+e y – the measurements for somatic cell counts β – the fixed effects of parity, stage of lactation, month of year u – random effect of cow, u ~ N (0, I δ2c ) e – random error, assuming e ~ N (0, I δ2e ) X, Z – incidence matrices for fixed effects and random cow effects respectively

RESULTS AND DISCUSSION The SCC significantly increased with advanced numbers of lactation. Especially group of cows on their fourth and higher lactations had 5.52±0.02 logx.ml-1 as compared with primiparous cows with 5.22±0.02 logx.ml-1. Cows on second lactation had 5.24±0.02 and cows on third 5.30±0.03 logx.ml-1. There were no significant differences among first three groups of parities indicating relatively low increase of health problems during first three lactations. In our earlier study (Tančin et al., 2007a) the multiparous cows had only numerically higher SCC as compared with primiparous cows and this difference is in agreement with other findings (Laevens et al., 1997). The clear increase of SCC from lactation to lactation was found in goat (Contreras et al., 1999). The months of the year (season) significantly influenced the SCC in dairy cows. There was a significant increase of SCC during the period of May, June and July as compared with winter months of the year (Fig. 1). Even during the June and July almost 18 % of cows had over million SCC in milk. Thus the summer time was critical for udder health in dairy farm. The higher SCC in our experiment suggested serious health problems of udders during summer period. It was reported that environmental pathogens caused higher incidence of mastitis during summer period (Smith et al., 1985) as a possible consequence of living conditions for bacteria (Mallet et al., 2012). Rupp and Boichard (2000) also claimed that the risk of first clinical mastitis was the highest around the second calving in lactation starting in summer period. Under the conditions of healthy mammary glands the season was pointed out to have no significant influence on SCC (Malinowski, 2001). More important factor is udder hygiene as pointed by Tongeľ and Brouček (2010). Above mentioned authors calculated positive coefficients of correlation between udder hygiene scores and mastitis at the level 0.77.

Slovak J. Anim. Sci., 46, 2013 (1): 31-34

Least squares means without a common superscript letter were significantly different at P <0.05 a–c

Fig. 1: The effect of season on SCC in the milk of dairy cows

Somatic cell count significantly changed throughout lactation. The lowest value was measured at second month and the highest at tenth month of lactation. As shown in Fig. 2 the first month of lactation was characterized by higher SCC as compared with second one. Under the practical conditions the periods after calving and the end of lactation are generally considered as critical for udder health. In our work we found the increase of SCC throughout lactation (Tančin et al., 2007a). Significant effect of the stage of lactation in dairy cows was also documented by Laevens et al. (1997). From the management point of view the period early postpartum and before drying are critical for udder health and dairy practice should take more care on cows during above mentioned periods of lactation.

Original paper In total during the experimental period 120 cows were calved in the herd. From them 55 % of cows were in low and 15 % in medium group. On the other side 80 cows were dried and 69 % of them were in low (38 %) and medium (31 %) groups. During both drying and calving period there were around 30 % of cows in a group of high. Though after calving the percentage of cows in low group increases (55 % versus 38 %), the high percentage of cows in high group indicate problems in the farm with the process of drying and calving. The dry therapy with antibiotics reduces the rate of udder infection in early dry period, but there was no effect during the prepartum period (Smith et al., 1985). Also above mentioned authors pointed out that dry cow therapy did not influence the incidence of environmental infection. Therefore the dry therapy with antibiotics is not the single solution to keep udder healthy as farmers initially thought. There are many other critical factors involved in higher risk of mastitis incidences during dry and transient period that should be taken into account in daily managing of dairy herd. CONCLUSION The season and stage of lactation significantly influenced SCC in milk of dairy cows under practical conditions. Both factors should be considered as a risk for mastitis and therefore should be taken into account in daily managing of dairy herd.

Acknowledgement This study was funded by the Operational Programme for Research and Development project “MLIEKO 26220220098” of the European Regional Development Fund.

REFERENCES

Least squares means without a common superscript letter were significantly different at P <0.05 a–b

Fig. 2: The effect of stage of lactation on SCC in the milk of dairy cows

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Original paper vol. 87, 2004, p.1256-1264. HOGEVEEN, H. – MILTENBURG, J. D. – Den HOLLANDER, S. – FRANKENA, K. 2001. Milking three times a day and its effect on udder health and production, IDF Mastitis Newsletter, vol. 24, 2001, p. 7. KELLY, A. L. – McSWEENEY, P. L. H. 2002. Indigenous proteinases in milk. Advanced Dairy Chemistry, vol. 1, 2002, p. 494-519. KUBICOVÁ, Ľ. – DOBÁK, D. 2012. [The development and the level of milk and milk product consumption in Slovak Republic and modulation of food demand in selected groups of families]. SPU Nitra, monograph, 2012, p. 88, ISBN 978-80-552-0821. LAEVENS, H. – DELUYKER, H. – SCHUKKEN, Y. H. – DE MEULEMEESTER, L. – VANDERMEERSCH, R. – DE MUÊLENAERE, E. – DE KRUIF, A. 1997. Influence of parity and stage of lactation on the somatic cell count in bacteriologically negative dairy cows. J. Dairy Sci., vol. 80, 1997, p. 3219-3226. LARRY SMITH, K. – TODHUNTER, D. A. – SCHOENBERGER, P. S. 1985. Environmental mastitis: Cause, Prevalence, Prevention. J. Dairy Sci., vol.68, 1985, p. 1531-1553. LEITNER, G. – CHAFFER, M. – SHAMAY, A. – SHAPIRO, F. – MERIN, U. – EZRA, E. – SARAN, A. – SILANIKOVE, N. 2004. Changes in milk composition as affected by subclinical mastitis in sheep. J. Dairy Sci., vol. 87, 2004, p. 46-52. MALINOWSKI, E. 2001, Somatic cells in milk. Medycyna Weterynaryjna, vol. 57, p. 13-17. MALLET, A. – GUÉGUEN, M. – KAUFFMANN, F. – CHESNEAU, CH. – SESBOUÉ A. – DESMASURES, N. 2012. Quantitative and qualitative microbial analysis of raw milk reveals substantial diversity

Slovak J. Anim. Sci., 46, 2013 (1):31-34 influenced by herd management practice. Int. Dairy J., vol. 27, 2012, p. 13-21. O´BRIEN, B. – MEANEY, W. J. – McDONAGH, D. – KELLY, A. 2001. Influence of somatic cell count and storage interval on composition and processing characteristics of milk from cows in late lactation. Australian J. Dairy Technol., vol. 56, 2001, p. 213218. PYÖRÄRLÄ, S. 2003. Indicators of inflammation in the diagnosis of mastitis. Veterinary Res., vol. 34, 2003, p. 565-578. RUPP, R. – BOICHARD, D. 2000. Relationship of early first lactation somatic cell count with risk of subsequent first clinical mastitis. Livestock Prod. Sci., vol. 62, 2000, p. 169-180. SANTOS, M. V. – MA, Y. – BARBANO, D. M. 2003. Effect of somatic cell count on proteolysis and lipolysis in pasteurized fluid milk during shelf-life storage. J. Dairy Sci., vol. 86, 2003, 2003, p. 2491. TANČIN, V. – IPEMA, A. H. – HOGEWERF, P. 2007. Interaction of somatic cell count and quarter milk flow patterns. J. Dairy Sci., vol. 90, 2007a, p. 22232228. TANČIN, V. – IPEMA, I. H. – HOGEWERF, P. H. – MAČUHOVÁ, J.: Sources of variation in milk flow characteristics at udder and quarter levels. J. Dairy Sci., vol. 89, 2006, p. 978-988. TANČIN, V. – UHRINČAŤ, M. – MAČUHOVÁ, L. – BRUCKMAIER, R. M.: Effect of pre-stimulation on milk flow pattern and distribution of milk constituents at a quarter level. Czech J. Anim. Sci., vol. 52, 2007b, p. 117-121. TONGEĽ. P. – BROUČEK, J. 2010 Influence of hygienic condition on prevalence of mastitis and lameness in dairy cows. Slovak J. Anim. Sci., vol. 43, 2010. p. 95-99.

ANALYSIS OF FATTENING ABILITY, CARCASS AND MEAT QUALITY OF HEAVY TSIGAI LAMBS

P. POLáK, J. TOMKA, E. KRUPA, K. ZAUJEC, Z. KRUPOVÁ, M. ORAVCOVÁ, J. HUBA Animal Production Research Centre Nitra, Slovak Republic

ABSTRACT The aim of the investigation was to analyze fattening ability, carcass and consumer meat quality from purebred Tsigai heavy lambs produced by sucking their mothers and natural grazing. Carcass and meat quality characteristics of 20 heavy lambs of Tsigai sheep were recorded at the average age of 137 days. Samples of musculus longissimus thoracis et lumborum were taken for physic-chemical analysis (proteins, fat, total water content) and consumer meat quality (pH, water holding capacity, electro conductivity, colour and shear force) 48 hours after slaughter. The devices Nicolet 6700 Spectrometer, Infratec 1265, pH meter Toledo and MiniScan XE plus or Spectrometer CM 2600 were used for the analysis. Seven days after slaughter, the grilling losses and shear force of grilled meat were obtained. Relatively high variability, more than 10 %, was found for dressing percentage, weight of carcass, weight of meat in carcass, weight of valuable cuts in carcass, average daily gain and lean meat production per day. The highest variability was found for fat proportion in carcass. Linear correlation coefficient was r = -0.41 for shear force and dressing percentage. On the other hand correlation coefficient of shear force with age and weight at slaughter were almost the same r = 0.40. It means that share force was increasing with increase in slaughter age and weight but decreasing with increase in dressing percentage. Despite of limited number of analysed animals in this preliminary study, these findings indicate that dual purpose breed Tsigai in the ewe-lamb production system is able to produce heavy lambs with good meat quality. Results also demonstrate a high variability within group of purebred Tsigai animals. Key words: sheep production; heavy lambs; Tsigai; meat quality

INTRODUCTION Currently sheep production is being one of very limited number of growing commodities in Slovak animal production, the second one is suckle cow production and meat sheep production. All commodities are closely connected to using natural sources of green matter by grazing. It is maybe due to quite suitable geographical and climatic conditions. More than 60 % of Slovak territory is hilly and mountain area with high level of steepness, high latitude, less fertile soil and high rainfall. More than 800 thousand hectares of natural meadows and pastures are located here. Sheep production is one of very traditional ways of animal production of mentioned hilly and mountain regions. There are two traditional breeds

*Correspondence: E-mail: polak@cvzv.sk Peter Polák, Animal Production Research Centre Nitra, Hlohovecká 2, 951 41 Lužianky, Slovak Republic Tel.: +421 37 6546 428 Fax: +421 37 6546 361

of sheep in Slovakia: Improved Valachian and Tsigai sheep. Traditional product of dairy sheep is fresh cheese from which the national speciality “Slovenská bryndza” a recognised product - has been prepared. Tsigai is a dual purpose breed kept in traditional milk production scheme named “Carpathian system” with lambing in winter and selling of light lambs before Easter. Breeding season is accommodated to the date of next year Easter, because majority of lambs should be sold at this event, at about 12-15 kg of live weight. After selling of lambs, the milking period starts and cheese is produced. Ewes are kept in mountain cottages named “salaš” during summer season. Production of heavy lambs by sucking their mothers on pastures is not traditional for the breed in the region, but due to various impacts, as diversification of

Received: February 29, 2012 Accepted: November 30, 2012

Original paper

Slovak J. Anim. Sci., 46, 2013 (1):35-38

production, market demands high price of specialised meat production breeding stock and lack of trained manpower is going to be more interesting today. The aim of the investigation was to analyze fattening ability, carcass and consumer meat quality from purebred Tsigai heavy lambs produced in the ewe-lamb production system by sucking their mothers and natural grazing.

MATERIAL AND METHODS Fattening characteristics, carcass and meat quality of 20 heavy lambs of Tsigai sheep were obtained at the average age of 137 days. Detail dissection of right half carcass was done 24 hours after slaughtering to obtain weight and proportion of basic tissues (muscle, fat and bones). Weight of valuable cuts was calculated as a sum of weight of round (boneless round without back shank), shoulder (boneless shoulder without front shank), back (boneless musculus longissimus thoracis et lumborum between 1st thoracic vertebra and last lumbar vertebra) and tender loin. The samples (approximately 500 g) were taken from MLTL (musculus longissimus thoracis et lumborum) and for the first time they were analysed as a fresh meat for physic-chemical meat quality after 48 hours of chilling. The samples of lamb’s meat were processed before the analysis (removal of surface fat, membranes and tendons) and grounded. Chemical parameters of meat (proteins, fat and total water content) were analysed afterwards, when no more changes in chemical composition of meat are in progress. The devices Nicolet 6700 Spectrometer or Infratec 1265 with the application module for fat content assessment 1 - 10 % were used. The pH meter Toledo with combined stab electrode was used to measure pH value. Water holding capacity was analysed by the Gramm Hama method. Meat colour (values L, a and b) were measured by the MiniScan XE plus and CM 2600 Spectrometer. Seven days after slaughter, when boneless meat is supposed to be in consumer maturity, the samples were

cut into approximately 2 cm slices and were grilled afterwards. Smaller samples, typical for lamb’s meat, were left untouched and grilled as a one piece. Contact grill PM-1015 was used for samples processing. After 4 minutes, grilling losses and shear force of grilled meat were measured by Texture Analyser. Grilling losses were valued by weighing the samples before and after grilling. Basic statistics of all obtained variables and correlation coefficients between selected characteristics of carcass and meat quality were calculated using SAS 9.2 statistical package.

RESULTS AND DISCUSSION Relatively high variability, more than 10 %, was found for dressing percentage, weight of carcass, weight of meat in carcass, weight of valuable cuts in carcass, average daily gain and lean meat production per day. The highest variability was found for fat proportion in carcass (Tables 1 and 2). Characteristics of consumer quality also showed high level of variability (Table 3). For example, shear force varied between 9.22 kg and 1.61 kg with average of 3.98 kg, what means that one piece of meat was very tender and the other one was too tough. For beef meat, acceptable level is between 4 kg and 6 kg. It could be said that our sample of heavy Tsigai lambs showed better results than those required for cattle, even slightly higher than light lambs` meat. It is probably related to the age of analysed lambs, which varied within 25 days, and live weight before slaughter (Table 4). In our opinion in the future we should focus on intramuscular fat level, level of daily gain and body/carcass conformation. It is very difficult to compare our results with results of other authors due to differences in breeds, sex, and age at slaughter or fattening strategies (Dickerson et al., 1972; Salomon et al., 1980; Nottern et al., 1991; Ochodnický et al., 1994; Brady et al., 2003; Daraban, 2008; Santos et al., 2008). Those authors, who analysed younger lambs (Ghita et al., 2009), lighter ones refer results about quality of very young lamb meat. As a

Table 1: Basic statistics of fattening performance of 20 Tsigai heavy lambs

Variable

Live weight before slaughter

Age at slaughter

Unit

Mean

Minimum

Maximum

Std Dev

32.95

29.00

42.00

3.10

days

137.40

123.00

151.00

8.27

Average daily gain

240.69

207.14

289.66

27.5

Weight of hot carcass

12.35

9.70

17.00

1.54

Dressing percentage

37.63

29.39

46.67

4.56

Lean meat yield per day

36.05

26.79

50.45

5.10

Slovak J. Anim. Sci., 46, 2013 (1): 35-38

Original paper

conclusion, it is possible to say that water content was higher and content of basic organic matter in the muscle was lower. Also shear force was lower for younger lamb meat. Apolen et al. (2002) refer similar results of carcass quality in their work; the only exception was fat content

in carcass, which was slightly higher. It is because they worked with a similar genotype; the only difference was the system of fattening. They applied common fattening based on conserved roughage and concentrates.

Table 2: �� Basic statistics of carcass quality �� characteristics of 20 Tsigai heavy lambs

Variable

Unit

Mean

Minimum

Maximum

Std Dev

Weight of meat in carcass

4.93

3.75

6.66

0.61

Weight of valuable cuts in carcass

2.90

2.25

4.12

0.38

Proportion of meat in carcass

70.44

68.16

75.40

2.4

Proportion of valuable cuts in carcass

41.45

38.58

44.69

1.30

Proportion of valuable cuts in meat

58.86

55.81

61.86

1.60

Proportion of bones in carcass

26.85

21.93

30.51

1.95

Proportion of fat in carcass

2.42

0.55

4.17

0.89

Table 3: Basic statistics of lamb meat quality characteristics of 20 Tsigai heavy lambs

Variable

Unit

Mean

Minimum

Maximum

Std Dev

Total water content

(g/100g)

76.24

74.80

77.90

0.75

Content of proteins

(g/100g)

20.65

20.10

21.30

0.33

Content of fat

Energetic value

pH after 48 hours

5.49

5.38

5.78

0.11

Electrical conductivity

(μS)

1.40

0.46

3.12

0.67

Colour L

(g/100g)

2.12

1.00

3.20

0.65

(KJ/100g)

425.68

374.35

472.33

25.40

40.77

35.98

44.45

2.03

7.57

5.81

9.19

0.98

8.51

5.94

9.97

1.01

36.67

25.27

44.97

6.06

Water holding capacity

(g/100g)

Grilling losses

(g/100g)

4.25

2.23

7.22

1.42

Shear force after 7 days

(kg)

3.98

1.61

9.22

2.00

Table 4: Correlation coefficients between selected characteristics of carcass and meat quality

Intramuscular fat Grilling losses content

Shear force after 7 days

Live weight before slaughter

0.36973

0.00350

0.40988

Age at slaughter

-0.10665

-0.12357

0.39630

Dressing percentage

-0.42331

0.11585

-0.41175

Proportion of fat in carcass

-0.20166

-0.19955

0.01295

Original paper CONCLUSIONS Despite of limited number of analysed animals in this preliminary study, these findings indicate that dual purpose breed Tsigai in the ewe-lamb production system is able to produce heavy lambs with good meat quality. Results also showed high variability of carcass and meat production traits within group of purebred Tsigai animals. Correlation analysis showed that shear force with age and weight at slaughter were almost the same r = 0.40. It means that shear force increasing with increase in slaughter age and weight at slaughter but decreases with increase in dressing percentage. In our opinion, we should focus to increase level of daily gain and body/carcass conformation in order to produce lamb meet with better consumer quality expressed in shear force of grilled meat.

ACKNOWLEDGEMENTS This article was written during realization of projects LAGEZ No. 26220120051 and CEGEZ No. 26220120042 supported by the Operational Programme Research and Development funded from the European Regional Development Fund. This work was supported by the Slovak Research and Development Agency under the contract no. APVV-0458-10.

REFERENCES APOLEN, D. – ČAPISTRÁK, T. – MARGETÍN, M. – MARGETÍNOVÁ, J. – ŠPÁNIK, J. – BLANCO ROA, N. E. 2002. Jatočná hodnota jahniat posudzovaná pomocou sonografu in vivo na základe jatočnej rozrábky a podľa zatriedenia EUROP. Vedecké práce VÚŽV Nitra, vol. 35, 2002, p. 149-156.

Slovak J. Anim. Sci., 46, 2013 (1):35-38 BRADY, A. S. – BELK, K. E. – LEVALLEY, S. B. – DALSTED, N. L. – SCANGA, J. A. – TATUM, J. D. – SMITH, G. C. 2003. An evaluation of the lamb vision system as a predictor of lamb carcass red meat yield percentage. J. Anim. Sci., vol. 81, 2003, p.1488-1498. DĂRĂBAN, S. 2008. Study concerning some carcass traits in young sheep fattened in different systems. Bulletin UASVM Animal Science and Biotechnologies, vol. 65 (1-2), 2008, p. 161. DICKERSON, G. E. GLIMP, H. A. – TUMA, H. J. – GREGORY, K. E. 1972. Genetic resources for efficient meat production in sheep growth and carcass characteristics of ram lambs of seven breeds. J. Anim. Sci., vol.34, 1972, p. 940-951. GHITĂ, E. – PELMUS, R. – LAZĂR, C. – REBEDEA, M. 2009. Comparative research on carcass quality in suckling lambs of different local sheep breeds. Archiva Zootechnica, vol. 12 (1), 2009, p. 38-47. NOTTER, D. R. – KELLY, R. F. – BERRY B. W. 1991. Effects of ewe breed and management system on efficiency of lamb production: III. Meat characteristics. J. Anim. Sci., vol. 69, 1991, p. 3523-3532. OCHODNICKÝ, K. – PALANSKÁ, O. – MARGETINOVÁ, J. 1994. Influence of live weight and age on chemical composition and quality of meat in lambs and ewes of Tsigai breed. Poľnohospodárstvo, vol. 40 (4), 1994, p. 272-281. SANTOS, V. A. C. – SILVA, S. R. – AZEVEDO, J. M. T. 2008. Carcass composition and meat quality of equally mature kids and lambs. J. Anim. Sci., vol. 86, 2008, p.1943-1950. SOLOMON, M. B. – KEMP, J. D. – MOODY, W. G. – ELY, D. G. – FOX, J. D. 1980. Effect of Breed and Slaughter Weight on Physical, Chemical and Organoleptic Properties of Lamb Carcasses. J. Anim. Sci., vol. 51, 1980, p.1102-1107.

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Slovak J.Anim.Sci., 46, 2013 (1) Contents

Original papers OLFATI, A. – MOGHADDAM, G.: Effects of GnRH agonist (CinnaRelin) on reproductive performance in synchronized Iranian crossbred ewes during the breeding season

CHRENEK, P. – MAKAREVICH, A. – KOZELOVÁ, D. – HIRIPI, L. – BOZSE, Zs.: Comparison of milk quality of transgenic rabbits carrying different gene constructs

SLAMEČKA, J, jr. – CHRENEK, P.�: Rabbit amniotic fluid as a potential alternative source of broadly multipotent stem cells

BOUSSENA, S. – BOUAZIZ, O. – DEHIMI, M. L. – HIRECHE, S. – AIMEUR, R. – KABOUIA, R.: The effects of electroejaculation on some physiological parameters (rectal temperature, respiratory and cardiac rates) in Ouled Djellal breed

AMOLE, T. A. – ODUGUWA, B. O. – SHITTU, O. – FAMAKINDE, A. – OKWELUM, N. – OJO, V. O. A. – DELE, P. A. – IDOWU, O. J. – OGUNLOLU, B. – ADEBIYI, A. O.: Herbage yield and quality of Lablab Purpureus during the late dry season in Western Nigeria

�� 22

TANČIN, V.: Somatic cell counts in milk of dairy cows under practical conditions

POLÁK, P. – TOMKA, J. – KRUPA, E. – ZAUJEC, K. – KRUPOVÁ, Z. – ORAVCOVÁ, M. – HUBA, J.: Analysis of fattening ability, carcass and meat quality of heavy Tsigai lambs

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