slovak journal of animal science, volume 45, 2012 (4) by CVŽV Nitra

Number

2012 Volume 45 45 (4) 111-147 ISSN 1337-9984

Slovak Journal of

Animal Science ANIMAL PRODUCTION RESEARCH CENTRE NITRA

Slovak Journal of Animal Science

Formely Journal of Farm Animal Science

Aims and scope

Slovak Journal of Animal Science (ISSN 1337-9984) is an international scientific Editor-in-chief: journal that publishes original Ladislav Hetényi, Animal Production Research Centre Nitra, Slovak Republic scientific papers, reviews, Executive editor: short communications, Ludmila Hanuliaková, Animal Production Research Centre Nitra, Slovak Republic chronicles of important Technical editor: jubilees, reports of Marta Vargová, Animal Production Research Centre Nitra, Slovak Republic participation in important international conferences on animal science in English Editorial board language. Topic of the journal Daniel Bíro, Slovak University of Agriculture Nitra, Slovakia are problems of animal Zsuzsanna Bosze, Agricultural Biotechnology Center, Gödöllö, Hungary production, mainly in the Jan Brouček, Animal Production Research Centre Nitra, Slovakia sphere of genetics, breeding, Jozef Bulla, Slovak University of Agriculture Nitra, Slovakia nutrition and feeding, Ondrej Debrecéni, Slovak University of Agriculture Nitra, Slovakia physiological processes Andrzej Filistowicz, The Faculty of Biology and Animal Science, University of digestion, conservation of Enviromental and Life Science, Wroclaw, Poland and treatment of feeds, Roland Grossmann, Institute of Animal Science Mariensee, Germany biotechnology, reproduction, ethology, ecologization Peter Chrenek, Animal Production Research Centre Nitra, Slovakia of breeding, biology and Jozef Laurinčík, Constantine the Philosopher University Nitra, Slovakia breeding technology in farm Juraj Koppel, Institute of Animal Physiology SAS, Košice, Slovakia animals, quality of meat, Peter Massanyi, Slovak University of Agriculture Nitra, Slovakia milk, wool, economy Gábor Mészáros, University of Natural Resouces and Life Sciences, of breeding and production Division of Livestock Sciences, Vienna, Austria of farm animals, mainly: Štefan Mihina, Animal Production Research Centre Nitra, Slovakia cattle, pigs, sheep, goats, Shoukhart M.Mitalipov, Oregon Health & Science University, Beaverton, U.S.A. horses, poultry, small farm Jaana Peippo, MTT Agrifood Research Finland, Jokioinen, Finland animals and farm game. Dana Peškovičová, Animal Production Research Centre Nitra, Slovakia There are published also articles from the sphere Juraj Pivko, Animal Production Research Centre Nitra, Slovakia Josef Přibyl, Research Institute for Animal production, Praha – Uhříněves, Czech Republic of biochemistry, genetics, embryology, applied Ján Rafay, Animal Production Research Centre Nitra, Slovakia mathematical statistics as Alexander Sirotkin, Animal Production Research Centre Nitra, Slovakia well as economy of animal Pavel Suchý, Department of Nutrition, Dietetics, Zoohygiene and Plant Products, production. There can be University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic published also articles from Milan Šimko, Slovak University of Agriculture Nitra, Slovakia the sphere of veterinary Peter Šútovský, University of Missouri – Columbia, U.S.A. medicine concerning the Vladimír Tančin, Animal Production Research Centre Nitra, Slovakia themes of the journal.

Editorial office

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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, 2012.

GENEALOGICAL ANALYSIS IN SMALL POPULATIONS: THE CASE OF FOUR SLOVAK BEEF CATTLE BREEDS

O. KADLEČÍK*, I. PAVLÍK Slovak University of Agriculture, Nitra, Slovak Republic

ABSTRACT The aim of the paper was to evaluate the genetic diversity in four beef cattle populations in Slovakia by the methods of pedigree analysis. The pedigree populations consisted of (reference populations in brackets) 481 (116) Blonde d`Aquitaine (BA) animals, 3,955 (1,772) Charolais (CH), 2,063 (786) Limousine (LI) and 916 (429) Simmental (SM). The highest inbreeding coefficient was in SM (3.87 %) and the lowest one in BA (0.33 %). Effective population size was computed via increase in inbreeding: 467.60 BA, 152.92 CH, 428.73 LI and 47.62 SM. The effective number of founders was 83, 233, 229 and 30 in BA, CH, LI and SM, respectively and the effective number of ancestors was 20, 72, 55, 13 in BA, CH, LI, SM, respectively. Contributions of animals to reference population were calculated. Unbalanced distribution of the most important ancestors within assessed populations was observed. The most significant genetic diversity loss was estimated in Simmental population. Key words: beef cattle; diversity; genetic diversity; inbreeding

INTRODUCTION Beef cattle breeding has not a long tradition in Slovakia. The very beginning of beef cattle breeding started in 70´s of the last century, when some Hereford animals were imported to former Czechoslovakia. These herds were not succesful and gradually disappeared. After 1990 few beef cattle breeds were imported to Slovakia (Charolais, Blonde d´Aquitaine, Limousine, Aberdeen Angus, Simmental, Piemontese, Highland, Galloway). The main goal of these imports was to create purebred populations and use these animals mainly in the program of suckling cows. In 1994 The Beef Cattle Breeders Association in Slovakia was established. This organization takes care about beef cattle development and it is responsible for breeding work and keeping of Herd Book as well. Slovak purebred beef cattle populations are small and their further development will depend on breeding programes of each breed. The breeding strategies currently applied in catlle breeding are effective in genetic gain generating. Efforts to improve genetic

*Correspondence: E-mail: ondrej.kadlecik@uniag.sk Ondrej Kadlečík, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic Tel.: 00421 37 6414291

level of breed lead to use a few superior animals. This advancement usually increases a probability of generation of inbred animals (Verrier et al., 1993). Therefore, it is very important to make breeding work purposefully because the long term survival of a population depends on the maintenance of sufficient genetic variation for individual fitness and population adaptability (Baumung and Sölkner, 2002). Maintaining of genetic diversity is a part of the goals in genetic management and evaluation of genetic diversity and knowing its level is the basis for effective genetic management of the breed. Genetic variability and its evaluation over time may be estimated from genealogical information. This methodology was used in more papers (Bozzi et al., 2006, Cervantes et al., 2009, Oravcová and Margetín, 2011, Kadlečík et al., 2012). The trend in inbreeding is the most frequently used to quantify the rate of genetic drift (Gutiérrez et al., 2003). The goal od this paper was to evaluate genetic diversity of four beef cattle breeds on the basis of their pedigree information describing parameters of the probability of identity by descent and gene origin.

Received: September 11, 2012 Accepted: November 9, 2012

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MATERIAL AND METHODS The analyzed pedigree populations consisted of (reference populations in brackets) 481 (116) Blonde d`Aquitaine (BA) animals; 3,955 (1,772) Charolais (CH); 2,063 (786) Limousine (LI) and 916 (429) Simmental (SM). Reference population (RP) set up heifers, cows and sires living in the year 2011. All animals were purebred and registered in Herd Books of The Beef Cattle Breeders Association in Slovakia. The analysis covered living sires (in insemination - AI and natural mating), as well as frozen genetic material deposited in reproduction centres. A total of 7,415 animals were registered. The number of animals according to each breed is presented in Table 1. The pedigree information was obtained from the database of The Beef Cattle Breeders Association in Slovakia. The software Endog v.4.8 (Gutiérrez and Goyache, 2005) was used for calculation of diversity parameters. At first, the pedigree completeness according to MacCluer et al., (1983) was evaluated. We used the index of pedigree completeness (PEC). PEC = 2 Csire Cdam / Csire + Cdam, where Csire and Cdam are contributions from the paternal d

and maternals lines. C = 1– ∑gi d

i=1

where gi is the

proportion of known ancestors in generation i; and d is the number of generations that are taken into account (Gutiérrez et al., 2009). The state of diversity was evaluated according to following parameters based on probability of identity by descent. The individual coefficient of inbreeding (F) reflects the probability that two individuals received two identical allels by descent (Gutiérrez et al., 2009). F value was computed according to algorithm of Meuwissen and Luo (1992). The average relatedness (AR) reflects the probability that an allele randomly chosen from the whole population in pedigree belongs to a given animal (Gutiérrez et al., 2009). The individual increase in inbreeding (∆Fi) was calculated by means of the classical formula ∆Fi = 1 - t-1√� 1 - Fi where Fi is individual

coefficient of inbreeding and t is the equivalent complete generation (Gutiérrez et al., 2009). We also calculated effective population size (Ne) defined as the number of breeding animals that would lead to actual increase in inbreeding if they contributed equally to the next generation (Gutiérrez et al., 2009). We assessed the parameters based on probability of gene origin, as well. Number of founders (f) where founder is animal with unknown genetic connections to other animals in pedigree except its own progeny (Lacy, 1989), was calculated. The effective number of founders (fe) defined as the number of equally contributing founders that will produce the same genetic diversity as assessed in the population (Boichard et al., 1997), was calculated as 1 fe = ∑kf = 1 qk2 where qk is the probability of gene origin of the k ancestor. The effective number of ancestors (fa) is the minimal number of ancestors necessary to explain the genetic diversity in the reference population (Boichard 1 et al., 1997), was calculated by formula fe = ∑aj = 1 qj2 where qj is the marginal contribution of an ancestor j.

RESULTS AND DISCUSSION The number of sires operating in artificial insemination and natural mating is presented in Figure 1. The higher percentage of bulls in natural mating goes with the pasture system of beef cattle breeding. The index of pedigree completeness (PEC) by the generations of ancestors for BA, CH, LI and SM is presented in Figures 2, 3, 4, 5, respectively. We can see that only BA and SM had 100 % completeness in the first generation of reference population. CH was very close to this level. Completeness had decreasing trend with increasing the number of generations. The lowest PEC through all generations was noted in LI. McParland et al., (2007) presented higher values of pedigree completeness in Irish beef cattle breeds (Charolais, Limousine,

Table 1: Description of analyzed beef breeds

Population

SM Total

116 1772

786

429 3103

Sex

304

482

217

212

481 3955 2063

Sex

229

252

707 1065 M

1532 2423

RP – reference population, PP – pedigree population, M – males, F – female

112

808 1255

1273 2011

916 7415

415

501

2984 4431

Slovak J. Anim. Sci., 45, 2012 (4): 111-117

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Simmental, Hereford and Angus). Very similar results to ours were presented by Bozzi et al., (2003) on original Italian beef breeds (Chianina, Marchigiana, Romagnola and Maremmana). The parameters of diversity based on probability of identity by descent are presented in Table 2. The highest inbreeding coefficient was in SM (3.87 %) and the lowest one in BA (0.33 %). The highest value of F was observed in SM (29.37 %). The highest average increase in inbreeding was in SM (1.05 %) as well. FAO (2007) states that average increase in inbreeding of one-half to one percent per generation is acceptable for maintaining of population. All observed populations are included in this range except SM. When we compare AR and F value, we can conclude that in the next periods an important growth of inbreeding may be expected. We calculated the effective population size via individual increase in inbreeding as well. The effective population size is an important parameter indicating the risk of population loss. The worst situation was observed in SM, where Ne was 47.62 individuals. The best situation was in BA

(Ne = 467.60) and LI (Ne = 428.73). GutiĂŠrrez et al., (2003) presented diversity parameters of eight Spanish beef breeds with a local importance. They found out that all eight breeds are endangered (Ne = 21 â&#x20AC;&#x201C; 123 individuals, increase in inbreeding by generation more than one percent in half of breeds).

Fig. 1: Number of bulls operating in AI and natural mating (ZCHMP, 2011)

Fig. 4: Pedigree completeness by the generations of ancestors in LI

Fig. 2: Pedigree completeness by the generations of ancestors in BA

Fig. 5: Pedigree completeness by the generations of ancestors in SM

Fig. 3: Pedigree completeness by the generations of ancestors in CH

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Table 2: Diversity parameters based on probability of identity by descent Parameter

PP n=481

x

0.33

0.14

1.05

0.47

0.35

1.05

0.97

3.10

2.14

1.54

Fi in %

xmin

xmax

7.03

12.50

25.10

25.00

29.37

x

2.58

1.25

1.00

0.55

1.14

0.62

6.65

3.53

0.90

0.98

0.51

0.57

0.60

0.61

2.84

3.62

RP PP n=786 n=2.063

RP n=116

RP PP n=1.772 n=3.955

RP n=429

PP n=916

0.14

3.87

1.90

1.01

5.19

4.17

AR in %

xmin

0.57

0.36

0.05

0.04

0.10

0.07

0.34

0.11

xmax

4.15

2.39

3.22

11.17

x

0.11

0.07

0.33

0.15

0.11

0.05

1.05

0.57

0.71

1.17

0.80

0.51

0.37

1.46

1.56

0.33

∆Fi in %

xmin

xmax

2.00

12.50

16.31

8.36

8.52

13.65

25.00

x

467.60

152.92

428.73

47.62

66.71

42.10

93.27

9.58

Ne via ∆Fi

Table 3: Diversity parameters based on probability of identity by descent – inbred animals Parameter

x s

RP n=26

PP n=29

RP n=431

PP n=435

RP n=109

PP n=117

RP n=271

PP n=277

1.49

2.31

4.33

4.31

2.53

2.55

6.13

6.28

1.81

3.32

5.04

5.02

3.41

3.45

5.37

5.49

Fi in %

xmin

0.05

0.04

0.15

0.10

0.39

xmax

7.03

12.50

25.10

25.00

29.37

x

3.21

3.02

1.33

1.70

1.68

8.38

8.26

0.46

0.72

0.36

0.37

1.07

1.41

AR in %

xmin

1.88

1.00

0.11

0.29

1.71

0.43

xmax

3.83

2.13

2.65

10.10

x

0.47

1.20

1.37

0.80

0.85

1.65

1.88

0.56

2.69

2.06

2.05

1.17

1.34

1.53

2.37

∆Fi in %

xmin

0.01

0.04

0.02

0.12

xmax

2.00

12.50

16.31

8.36

8.52

13.65

25.00

The ratio of inbred animals in RP was 22.4 % in BA, 24.32 % in CH, 13.87 % in LI and 63.17 % in SM. The results of parameters based on probability of identity by descent of inbred animals are presented in Table 3.

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The parameters based on probabilty of gene origin are presented in Table 4. We can see that fe value was 83, 233, 229 and 30 in BA, CH, LI and SM, respectively. The fa value was 20, 72, 55, 13 in BA, CH, LI, SM,

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respectively. A disbalance between fa and fe indicates bottleneck occurrence in pedigrees of all breeds. The same situation was observed in eight Spanish beef cattle breeds by Gutiérrez et al., (2003). A half of diversity was explained by lower number of ancestors in all breeds. McParland et al. (2006) presented very similar results in Irish beef cattle population. The fa was 58, 82,

35 in CH, LI and SM, respectively. Bozzi et al., (2003) presented higher values of parametres based on gene origin in Italian beef breeds but these populations were incomparably greater than ours. The contributions of the five most important ancestors are presented in Table 5. We can see unbalanced distribution of the most important ancestors within

Table 4: Diversity parameters based on probability of gene origin

Parameter

Number of founders (f)

191

1225

1219

786

763

429

674

Effective number of founders (fe)

136

233

381

229

324

Effective number of ancestors (fa)

139

123

Number of ancestors contributing to population

135

562

877

373

555

171

Number of ancestors explaining 50 % of diversity

Table 5: Contributions of the most important ancestors to reference population

Breed

Ancestors

sex

Marginal contributions

individual

cumulative

1 BA 2 3 4 5

BDV 051 BDV 509 BDV 081 3291132274 BDV 511

♂ ♂ ♂ ♀ ♂

13.04 9.48 7.01 6.08 6.04

13.04 22.52 29.53 35.61 41.65

1 CH 2 3 4 5

CHV 529 CHV 161 IL 000* CHV 241 CHV 171

♂ ♂ ♂ ♂ ♂

4.83 4.03 4.02 3.54 2.87

4.83 8.86 12.88 16.42 19.29

1 LI 2 3 4 5

LIV 508 ZLI 234 ZLI 207 ZLI 229 LIV 525

♂ ♂ ♂ ♂ ♂

7.48 4.91 4.06 3.66 3.59

7.48 12.39 16.45 20.11 23.70

1 SM 2 3 4 5

SBV 041 SIM 001 SIM 002 SBV 589 921377

♂ ♂ ♂ ♂ ♀

17.98 12.84 11.23 8.00 5.64

17.98 30.82 42.05 50.05 55.69

* - the bull has not got Slovak state register (name:FONTENA, HB number: 8690100259)

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assessed populations.The highest individual contribution was calculated in SM, where the sire SBV-041 (SWITZ HTF PANAMA RED) from USA had 17.98 %. The sire BDV-051 (DROP) from France was the most important ancestor in BA population with individual contribution of 13.04 %. The bull CHV-529 (CIKA-ET) born in Czech Republic was the most influential CH ancestor with contribution of 4.83 % what was the lowest top-

value among all breeds. The sire LIV-508 imported from France to Slovakia was the most important LI ancestor with individual contribution of 7.48 %. There were 2 cows in Top 5 ancestors in BA and SM. The trends of individual and cumulative contributions of 50 most important ancestors in BA, CH, LI and SM are presented in Figures 6, 7, 8 and 9, respectively.

Fig. 6: Cumulative and individual contributions of the most important ancestors in BA

Fig. 7: Cumulative and individual contributions of the most important ancestors in CH

Fig. 8: Cumulative and individual contributions of the most important ancestors in LI

Fig. 9: Cumulative and individual contributions of the most important ancestors in SM

CONCLUSION

Slovakia and unbalanced using of ancestors in reference populations. The level of inbreeding is not high except SM. The reduction of inbreeding should be based on optimization of mating programs and import of unrelated animals from abroad.

All populations assessed in this study are small and newly creating. They were based on imported animals (France, USA, Czech Republic, Hungary). The most significant genetic diversity loss can be observed in SM population. One of the most important factors reducing diversity in these populations is bottleneck effect and drift of genes. The reason for this state can be a disappearance of some important breeders and their animals which were the pioneers of beef cattle breeding in

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ACKNOWLEDGEMENTS The authors thank The Beef Cattle Breeders Association in Slovakia for providing pedigree

Slovak J. Anim. Sci., 45, 2012 (4): 111-117 information, especially Ing. Oľga Motolová for help with modification of databases. This work has been supported by the Excellence Centre for Agrobiodiversity Conservation and Benefit project ITMS 26220120015 implemented under the Operational Programme Research and Development financed by European Fund for Regional Development. This work has been supported by the Slovak Research and Development Agency under the contract No. APVV-0636-11 and Centre of excellence for preservation and utilization of genetic resources ECOVA No. 26220120015 and ECOVA plus No. 26220120032.

REFERENCES BAUMUNG, R. – SÖLKNER, J. 2002. Analysis of pedigrees of Tux-Zillertal, Carinthian Blond and Original Pinzgau cattle population in Austria. J. Animal. Breed. Genet., vol. 119, 2002, p. 175-181. BOICHARD, D. – MAIGNEL, L. – VERRIÉR, E. 1997. The value of using probabilities of gene origin to measure genetic variability in a population. Gen. Sel. Evol., vol. 29, 1997, p. 5-23. BOZZI, R. – SIRTORI, F. – FORABOSCO, F. – FRANCI, O. 2003. Pedigree analysis of Italian beef cattle breeds. Ital. J. Anim. Sci., vol. 2, 2003, p. 121123. BOZZI, R. – FRANCI, O. – FORABOSCO, F. – PUGLIESE, C. – CROVETTI, A. – FILIPPINI, F. 2006. Genetic variability in three Italian beef cattle breeds derived from pedigree information. Ital. J. Anim. Sci., vol. 5, 2006, p. 129-137. CERVANTES, I. – GUTIÉRREZ, J. P. – MOLINA, A. – GOYACHE, F. – VALERA, M. 2009. Genealogical analyses in open populations: the case of three Arabderived Spanish horse breeds. J. Anim. Breed. Genet., vol. 126, 2009, p. 335-347.

Original paper FAO UN. 2007. The state of world´s animal genetic resources for food and agriculture. Rome : FAO, 2007, 511 p. ISBN 978-92-5-105762-9. GUTIÉRREZ, J. P. – ALTARRIBA, J. – DÍAZ, C. – QUINTANILLA, R. – CAÑON, J. – PIEDREFITA, J. 2003. Pedigree analysis of eight Spanish beef cattle breeds. Genet. Sel. Evol., vol. 35, 2003, p. 43-63. GUTIÉRREZ, J. P. – GOYACHE, F. – CERVANTES, I. 2009. Endog v.4.6. A Computer Program for Monitoring Genetic Variability of Populations Using Pedigree Information. User´s Guide, 2009, p. 38 KADLEČÍK, O. – PAVLÍK,I. – HAZUCHOVÁ, E. – KASARDA, R. 2012. Assessing the genetic diversity in the Slovak Sport Pony using genealogic information. Acta Fytotechnica et Zootechnica, vol. 15 (1), 2012, p. 8-13. LACY, R. 1989. Analysis of Founder Representation in Pedigrees : Founder Equivalents and Founder Genome Equivalents. Zoo Biology, vol. 2, 1989, p. 111-123. MC PARLAND, S. – KEARNEY, J. F. – RATH, M. – BERRY, D. P. 2007. Inbreeding trends and pedigree analysis of Irish dairy and beef cattle populations. J. Anim. Sci., vol. 85, 2007, p. 322-331. ORAVCOVÁ, M. – MARGETÍN, M. 2011. Preliminary assessment of trends in inbreeding and average relatedness of the former Valachian sheep. Slovak J. Anim. Sci., vol. 44 (3), 2011, p. 90-96. VERRIER, É. – COLLEAU, J. J. – FOULLEY, J. L. 1993. Long term effects of selection based on the animal model BLUP in a finite population. Theor. Appl. Genet., vol. 87, 1993, p. 446-454.

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EFFICIENCY OF TREATMENT OF FOLLICULAR CYSTS IN COWS

D. ŠŤASTNÁ, P. ŠŤASTNÝ Slovak University of Agriculture, Nitra, Slovak Republic

ABSTRACT The aim of the work was to compare two protocols of treatment of cows with follicular cysts. 368 cows of Holstein bred were assigned for the experiment. They were 2-5 years old with average milk production of 9580 kg. The cows with ovarian cysts were randomly divided into two groups according to the treatment method. Diagnostics of the follicular cyst was performed by a transrectal ultrasonographic examination by 5 MHz linear probe. Group 1 consisted of cows (186) which were treated with GnRH on the 0 and 7th day ( 50µg, im; Supergestran ), PGF2α on 14th day (25mg, im; Oestrophan, Spofa), GnRH on 16th day (50µg, im). Insemination was done on the 17th day (20±4 hours after GnRH). Group 2 consisted of cows (182) treated with GnRH on 0 day (50 µg), PGF2α (25 mg) on 7th day and GnRH (50 µg) on 9th day. Insemination was done on the 10th day (20±4 hours after GnRH). The cows were ultrasonographically examined on the 0 and 7th day. On the 7th day after first treatment with GnRH better results (P< 0.05) were ascertained in the second group after identical treatment. In the subgroup 60 to 70 days post-partum presence of CL was of 4.54 % more and in the subgroup at 101-150 days post-partum the number of CL was up to 22.18 % more. This difference among the groups makes 28.99 %. The cows treated between 71st and 100th days post-partum in both groups had almost identical occurrence of preovulatory follicles. Significantly better efficiency (P<0.05) was achieved in the second group (31.32 %). More preovulatory follicles were ascertained at 60 to 70 days post-partum (up to 4.54 % more; P<0.05) and at 101-150 days (up to 22.18 % more). From the initial number of animals assigned for the treatment after insemination until 30 days only 30.65 and 36.65 % cows, respectively, remained pregnant. The most cows were concepted in the subgroup of 71-100 days of puerperium (15.59 % or 14.84 %). Both protocols of the treatment showed to be effective methods for the therapy of follicular cystic degeneration. Key words: cows; ovarian cysts; Ovsynch

INTRODUCTION Reproductive efficiency is one of the most important factors determining the profitability of the herd of dairy cows. Calving interval is the deciding parameter used for assessment of the reproductive efficiency. Oneyear calving interval is considered as economically advantageous as well as physiologically acceptable (Schmidt, 1989). Regular and sufficiently intensive puerperal ovarian activity, on-time display and estrus detection, properly scheduled insemination within 85 day peurperium despite the high production of milk are necessary for reaching such interval with breedingcows. Puerperium is a period of involutional processes of sex organs of a cow after parturition. According to Hajurka

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(2005) involution of the uterus of cows consists of three processes: from the contraction of myometrium, loss of tissue and shedding the surface of carunculs and renewal of the epithelium of endometrium. The period within 20-30 days after parturition is considered as physiological course of uterus involution (Doležel, 1989). Among the basic indicators of successfully proceeding puerperium in cows are non-malodorous lochie, end of its discharge up to 14 days after parturition, first puerperal estrus during the fourth week from parturition with following estrus in three week time and normal estrous mucus (Bouška et al., 2006). Effective puerperal estrus, in the period from 40th to 100th day after parturition, is in the herds with high milk production often a problem, which is associated with high occurrence of follicular cysts (6-30 %), while in

*Correspondence: E-mail: danka.stastna@uniag.sk; pavel.stastny@uniag.sk Danka Šťastná, Pavel Šťastný, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic Tel.: 00421 37 6414272, 00421 37 6414471

Received: July 3, 2012 Accepted: October 2, 2012

Slovak J. Anim. Sci., 45, 2012 (4): 118-122

Original paper

80 % of cows estrus is absent and in 20 % of cows estrus can be very intensive (Šťastný and Lacková, 2004). Follicular cysts in cows are hypertrophic follicles which do not ovulate in the estrus period (Garverick, 1999). Since these cows remain temporarily sterile they make an economic loss for the breeder. The loss depends on the frequency of occurrence of cysts. The exact cause of ovarian cysts is unknown, but the lack of hypothalamic gonadotropin – releasing hormone (GnRH) in the estrus period plays an important role in its pathogenesis (Gumen et al., 2003). Lack of GnRH can have various reasons, from genetic to alimentary and stress causing reasons. These can be peripartal disorders, milk fever, retention of placenta, endometritis, high content of oestrogens in the fodder, etc. The lack of endogenous GnRH is shown also by the positive reaction to the application of exogenous GnRH to cows with follicular cysts which induces ovulation of the follicle or luteinisation of the ovarian cyst (Fricke et al., 1998; Garverick, 1999, Stevenson, 2012) and stimulates new follicular wave. Its application to cows without ovarian cyst via new follicular wave can cause increased occurrence of follicle atresia (MacMillan and Thatcher, 1991). Protocol for synchronisation of ovulation and timing of insemination (Ovsynch), which can affect the percentage of gravidity if it is timed to the early luteal phase, is used for cows without ovarian cysts at a specific stage of estrous cycle (Pursley et al., 1997; Moreira et al., 2000). According to Ježková (2006) is it possible to avoid these problems by examination of the state of health in puerperium and by assessment of the quality of feed ration. Regular puerperal examination of the reproductive organs and thorough observation of the displays of estrus as well as usage of instrumental technique in clinical gynaecological practice can put this problem under effective control which could increase the possibilities of early cyst elimination. The aim of the work was to compare two protocols of treatment of cows with follicular cysts.

MATERIAL AND METHODS 368 cows of Holstein bred at the age from 2 to 5 years with average productivity of 9580 kg of milk in the period from April to August were assigned for the experiment. The cows were stabled freely in categories according to productivity. Gynaecological examination of cows was conducted in the herd in 14 day intervals since 60±7 days after parturition. Cows with follicle cysts were afterwards divided into two groups according to the method of treatment. Diagnostics of follicular cysts was conducted using transrectal ultrasonographic examination (DP-3300 Vet Digital Ultrasonic Imaging System, Midray Medical Corp. USA) with 5 MHz linear probe. As a criterion for cyst identification was the size of follicle (> 25mm), the wall thickness of the follicle and the absence of corpus luteum (CL). Diagnostics of gravidity was conducted following 30±3 days after insemination using ultrasonographic examination and following 60±5 days manually using rectal examination. The occurrence of follicular cysts of various forms after the parturition (< 70, 71-100, 101-150, > 150 days), the presence of corpus luteum (CL) seven days after treatment with GnRH and the presence of preovulatory follicles at the day of insemination were assessed statistically by the computational software of SAS. Pregnancy rate per AI was defined as the number of pregnant cows to total number of animals. The group 1 consisted of cows (186) treated with GnRH (50µg, im; Supergestran) on 0th and 7th day, PGF2α (25mg, im; Oestrophan Spofa) on 14th day and GnRH (50µg, im) on 16th day. Insemination was conducted on the 17th day (20±4 hours after the treatment with GnRH). The group 2 consisted of cows (182) treated with GnRH on 0th day (50 µg), PGF2α (25 mg) on 7th day and GnRH (50 µg) on the 9th day. Insemination was conducted on the 10th day (20±4 hours after treatment with by GnRH). The cows were ultrasonographically examined on the 0th day and 7th day.

Protocols of the treatment group 1 GnRH

GnRH

PGF2α

GnRH

Insemination

0 day3

7th day

14th day

16th day

17th day

GnRH

PGF2α

GnRH

Insemination

0 day

7th day

9th day

10th day

group 2

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Slovak J. Anim. Sci., 45, 2012 (4): 118-122

RESULTS AND DISCUSSION Follicular cysts were diagnosed in 368 cows. These were treated by two methods. The treatment in both groups started (0th day) by application of GnRH. The period from the treatment to insemination in the first group was 17 days and in the second group - 10 days. Within both groups the animals were divided into

subgroups according to the length of puerperal period when the ovarian cysts were detected. The results of presence of CL on 7th day after initial treatment with by GnRH are shown in the Table 1. Better results (P< 0.05) were ascertained in the second group, although the performed treatment was identical. There was 4.54 % more CL present in the subgroup of 60 - 70 days post-partum of the group 2. The difference in the second subgroup of 101-150 days post-partum

Table 1: Presence of CL on the 7th day after the treatment of cows with GnRH at different time of puerperium

CL/7th day

Days post partum

60 - 70

Group 1

n-186

Group 2

n 182

6.45

% 10.99a

71-100

20.97

21.43

101-150

9.14

31.32a

> 150

7.53

9.34

total

82/186

44.09

133/182

73.08a

a-P<0.05

Table 2: Presence of preovulatory follicles at the day of artificial insemination

Days post partum

Group 1

Group 2

n-186

n 182

60 - 70

5.9

10.44a

71-100

19.89

19.78

101-150

9.14

31.32a

> 150

4.83

6.59

total

74/186

39.78

124/182

68.13a

a-P<0.05

Table 3: Gravidity on the 30th day after artificial insemination

Days post partum

60 - 70

Group 1

n-186

Group 2

n 182

3.76

8.79

71-100

15.59

14.84

101-150

9.14

7.69

> 150

2.15

4.95

total

57/186

30.65

66/182

36.26

120

Slovak J. Anim. Sci., 45, 2012 (4): 118-122 between groups was 22.18 %. Total difference between the groups makes 28.99 %. Similar findings were reported by Hendricks (2004), who used the same protocols of treatment, which he rationalized with different levels of progesterone at the time when the cows were put into observation. These differences are probably resulting from random categorization of cows into groups. It could result in the fact that cows in the second group had more even and lower progesterone concentration or lower ovary sensitivity to GnRH. The tendency of CL presence is about the same, when the most CL were ascertained in the subgroup between 71st and 150th day, in the second group it was mainly after the 100th day of puerperium. The cows in both groups treated between the 71st and 100th day of puerperium showed almost identical occurrence of preovulatory follicles. As results from the Table 2, there is a difference in the efficiency among treatments presented by the presence of preovulatory follicles, which results from the differentiated treatment procedure since the 7th day after putting the cows into experiment. Significantly higher efficiency (P<0.05 ) was reached in the second group (31.32 %). Similarly to the CL presence, more preovulatory follicles (4.54 %) were ascertained in the subgroup of 60 to 70 days post-partum (P<0.05). It was at 22.18 % more in the group of 101-150 days post-partum. It is probable that the combination of application of PGF2α on the 7th day and GnRH on the 9th day was more effective also from the point of already mentioned individual sensitivity. According to Moreira et al. (2000), cows treated according to Ovsynch protocol in the early diestrum (group 2) respond to the treatment more intensively. The cows between 71st and 100th day post-partum responded less intensively than the cows between 101-150 days post-partum. This supports the assumption about the ovary sensitivity, since at that time the milk production culminated, which was probably the dominant factor (Lopez-Gatius et al., 2002). Despite stated significant differences in the ovarian reaction in both groups there were not ascertained significant differences from the point of treatment protocol in the gravidity of the animals on the 30th day at ultrasonographic examination (Tab.3). Thirty days after insemination there were 30.65 % and 36.26 % of pregnant cows from the initial number of treated animals. The most cows were concepted in the subgroup of 71-100 days of puerperium (15.59 % or 14.84 %). Both protocols of treatment turned out as effective methods of therapy of ovarian cyst degeneration. Higher efficiency is shown from the point of duration of the treatment at shortened protocol used in the second group (17 or 10 days), as well as from the point of manipulation with animals during their examination and treatment and from the point of cost of medication (GnRH was repeatedly applied on 7th day in the first group). Ascertained facts show the importance of the observation and on-time treatment of ovarian cystic

Original paper disorder, because their treatment after the culmination of lactation curve does not have significant effect on their treatment, but only decreases losses to the reproduction process which are caused by ovarian cysts (Gossen and Hoedemaker, 2006).

CONCLUSION Cystic ovarian disease in dairy cattle occurs most frequently during the post-partum period up to 70 days after calving. In conclusion, the results of this study suggest that fertility may not be different between cows with ovarian cysts treated with either the shorter or the longer Ovsynch protocol in this dairy herd.

ACKNOWLEDGEMENT The authors are grateful to the VEGA grant No. 1/9076/02. for the financial support of this study.

REFERENCES BOUŠKA, J. 2006. Chov dojeného skotu. Praha: Nakladatelství odborného tisku Profi Press, 2006. s. 96-162. ISBN 80-86726-16-9 DOLEŽEL, R. 1989. Dynamika změn vybratých ukazovatelů fyziologie puerperia na pohlavním ústrojí a v periferii krvi u krav. Brno: Vysoká škola veterinární, 1989, s 3-8. FRICKE, P. M. – GUENTHER, J. N. – WILTBANK M. C. 1998. Efficacy of decreasing the dose of GnRH used in a protocol for synchronization of ovulation and timed AI in lactating dairy cows. Theriogenology, vol. 50, 1998, p. 1275-1284. GARVERICK, H. A. 1997. Ovarian follicular cysts in dairy cows, J. Dairy Sci.,vol. 80, 1997, p. 995-1004. GARVERICK, H. A. 1999. Ovarian follicular dynamics and endocrine profiles in cows with ovarian follicular cysts. In: HOWARD J. L., SMITH R. A., editors. Current Veterinary Therapy, Food Animal Practice. WB Saunders Company, 1999, p. 577-587. GOSSEN, N. – HOEDEMAKER, M. 2006. Reproductive performance of dairy cows with relation to time of ovarian cyst formation, Bull. Vet. Inst. Pulawy, vol. 50, 2006, p. 159-161. GÜMEN, A. – GUENTHER J.N. – WILTBANK, M. C. 2003. Follicular size and response to Ovsynch versus detection of estrus in anovular and ovular lactating dairy cows. J Dairy Sci., vol. 86, 2003, p. 3184-3194. HAJURKA, J. 2005. Možnosti ovplyvňovania involúcie maternice. Infovet, roč. 12, č. 4, s. 181-184.

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Original paper HENDRICKS, K. E. M. 2004. Reproductive strategies in the postpartum dairy cow with reference to anovulation and postpartum uterine health. A Thesis presented to the graduate school, University of Florida, 2004. MOREIRA, F. – DE LA SOTA, R. L. – DIAZ, T. – THATCHER, W. W. 2000. Effect of day of the estrous cycle at the initiation of a timed artificial insemination protocol on reproductive responses in dairy heifers. J. Anim. Sci., vol. 78, 2000, p. 15681576. LOPEZ-GATIUS, F. – SANTOLARIA, P. – ZANIS, J. – FENECH, M. – LOPEZ-BEJAR, M. 2002. Risk factors for postpartum ovarian cyst and their spontaneous recovery or persistence in lacting dairy cows. Teriogenology, vol. 58, 2002, p. 1623-1632.

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Slovak J. Anim. Sci., 45, 2012 (4): 118-122 PURSLEY, J. R. – KOSOROK, M. R. – WILTBANK, M. C. 1997. Reproductive management of lactating dairy cows using synchronization of ovulation. J. Dairy Sci., vol. 80, 1997, p. 301-306. SCHMIDT, G. H. 1989 Effect of lenght of calving intervals on income over feed and variable costs. J. Dairy Sci., vol. 72, 1989, p. 1605-1611. STEVENSON, J. 2012. Eleven truths about ovarian cysts. Hoard‘s Dairyman, The National Dairy Farm Magazine, January, 2012, p. 21. ŠŤASTNÝ, P. – LACKOVÁ, D. 2004. Praktická reprodukcia zvierat - hovädzí dobytok. Nitra. 2004. s. 66-88. ISBN 80-969232-4-2.

EFFECT OF IN VITRO CULTURE CONDITIONS ON THE QUALITY OF RABBIT EMBRYOS

B. KULÍKOVÁ3, J. SLAMEČKA JR.1,2, A.V. MAKAREVICH1, P. CHRENEK1,2* Animal Production Research Centre Nitra, Slovak Republic Slovak University of Agriculture, Nitra, Slovak Republic 3 Constantine the Philosopher University, Nitra, Slovak Republic 1 2

ABSTRACT The aim of our study was to examine effect of in vitro culture on preimplantation rabbit embryo quality (total cell number, number of blastomeres in the inner cell mass (ICM) area, apoptotic index and embryo diameter). Rabbit embryos of the New Zeland White breed were flushed from the oviducts 19 – 20 hours post coitum and cultured up to the blastocyst stage (the experimental group). As a control group, the embryos developed in vivo were used, which were flushed from the uterus at the expanded blastocyst stage 94 hours post coitum. Embryos were stained by propidium iodide and Hoechst 33342 to detect total cell number and number of cells in the ICM area. For detection of apoptotic cells, TUNEL-reaction was used. Significantly higher number of blastomeres in ICM (p<0.05) was found out in the control group (44.18±2.50) when compared to the experimental group (36.22±2.45). However, total cell number of embryos was not significantly different between these two groups (155.12±15.00 vs. 127.23±14.50), respectively. Significant difference between control and experimental groups in the number of apoptotic cells (0.35 vs. 1.25) and embryo diameter (133.95±8.95 vs. 123.40±7.25 μm) was not proved too. Our results demonstrate slightly worse quality of rabbit embryos cultured in vitro, compared to embryos developed in vivo what is reflected in the number of cells in embryoblast. Key words: rabbit; embryos; in vitro cultivation; embryo quality

INTRODUCTION The most useful criterium of quality of preimplantation embryos is the evaluation of developmental stage of the embryos basing on visual inspection of their morphology under a light microscope. However, such evaluation is often confused and subjectively influenced. In the earlier report the embryo cell number was proposed as a valid indicator of embryo quality (Papaioannou and Ebert, 1988). Cell number of bovine in vitro produced blastocysts varied depending on the morphological grade, and later developing blastocysts were of poor quality as proved by the cell number (Jiang et al., 1992). It was observed that embryos developing quickly to the blastocyst stage had a higher total cell

*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

number than embryos developing slower (Iwasaki et al., 1990). The developmental potential of a single blastomere from mammalian embryo is usually determined by its ability to form blastocyst with a visible inner cell mass (ICM) and a distinct trophoblast (Chrenek et al., 2008). For a better estimation of embryo quality and allocation of cells to the embryoblast and the trophoblast, differential staining can be used. The possibility of using this technique enables a good comparison of ICM development under different culture conditions and to control for deviations in development due to micromanipulations or environmental influences (Van Soom et al., 2001). The proportion of apoptotic cells has been considered one of the most important

Received: October 25, 2012 Accepted: November 20, 2012

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Slovak J. Anim. Sci., 45, 2012 (4): 123-126

parameters for evaluating embryo viability or culture conditions (Chrenek and Makarevich, 2005; Makarevich et al., 2006). Another non-invasive parameter for the testing of embryo viability seems to be an embryo diameter. Mori et al. (2002) reported high positive correlations between the cell number and the diameter of bovine embryos collected on days 7-9 after in vitro fertilization. The measurement of embryo diameter and cell number using nuclear staining with vital permeant dyes (Hoechst 33342, DAPI) enables the non-invasive selection of the embryos with best developmental potential without its destruction (Makarevich et al., 2005). The aim of this study was to evaluate quality of the preimplatation rabbit embryos fertilized in vivo and developed either under conditions of the maternal tract or in vitro conditions, examining the total cell number, ICM cell number and the diameter of rabbit embryos as well as number of apoptotic cells.

MATERIAL AND METHODS Biological material Three days before mating, New Zealand White rabbit donors (APRC Nitra, SR) were treated with PMSG (Werfaser, 20 IU/kg of body weight, Austria) followed by hCG (Werfachor, 40 IU/kg of body weight, Austria) 72 h later (Chrenek et al., 2002). At 19 to 20 hours post coitum (hpc), the pronuclear stage eggs (the experimental group) were flushed from the oviducts of the rabbit females with PBS (Sigma, USA). The selection of flushed eggs was done in CIM medium added with fetal bovine serum (FBS) (10 %; Gibco BRL, USA). Presumptive zygotes were cultured in vitro in k-DMEM medium with 10 % of fetal bovine serum (FBS; Gibco BRL, USA) up to the expanded blastocyst (ExBl) stage for 94 hours at 39°C in the atmosphere of 5 % CO2 in air.

Rabbit embryos flushed from the uterus of does 94 hpc at the expanded blastocyst stage were used as a control group. Differential staining The embryos were incubated in freshly prepared 0.2 % Triton X-100 in PBS containing 2 mg/ml BSA for 20 s, and immediately washed twice in PBS-BSA medium. The embryos were transferred into PBS-BSA containing 30 µg/ml of propidium iodide (PI) and incubated in the dark at 37°C in warm chamber for 5 min, and then washed twice in PBS-BSA medium. Next, the embryos were incubated in 4 % paraformaldehyde (PFA) containing 10 µg/ml bisbenzimide (Hoechst 33342, Sigma, USA) for 30 min at room temperature and then washed twice in PBS-BSA medium. The embryos were incubated in a freshly prepared ice-cold solution of 0.1 % Triton X-100 in 0.1 % sodium citrate (v/v) for 5 min and then washed twice in PBS-BSA medium (Fouladi-Nashta et al., 2005). Afterwards, the embryos were covered with 5 µl of Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA), attached to microslide using small columns of nail polish (Makarevich et al., 2005) and examined under a Zeiss confocal microscope. Analysis of apoptosis (TUNEL) The embryos were removed from culture medium, washed 3x5 min in PBS supplemented with polyvinylpyrrolidone (PBS-PVP, 4 mg/ml) and then fixed in 3.7 % formalin for 5 min and in 70 % ethanol for 10 min. For membrane permeabilization, the embryos were incubated in 0.5 % Triton X-100 in PBS for 15 min. The embryos were processed for TUNEL using a MEBSTAIN Direct Apoptosis Detection Kit (Immunotech, Marseilles, France) according to the manufacturer´s instructions. Afterwards, the embryos were counterstained with propidium iodide (PI, 1 µg/ml in PBS). After the three-time washing of embryos in PBSPVP, they were covered with 5 µl of Vectashield mounting

Table 1: Quality of rabbit embryos after in vitro culture No. of Diameter of No. of cells in Group ExBl embryos (mm) Total cell no. ICM ICM/total (%)

x ± SD

(N) x ± SD

x ± SD

control 36 133.95±8.95a 155.12±15.00a 44.18±2.50a (28.48) in vitro 45 123.40±7.25a 127.23±14.50a 36.22±2.45b (28.46) cultured

vsb significant different at p<0.05

124

No. of apoptotic cells/embryo

x ± SD (%) 0.35±0.25a (0.25 %) 1.25±1.50a (1.00 %)

Slovak J. Anim. Sci., 45, 2012 (4): 123-126 medium (Vector Laboratories, Burlingame, CA, USA) and attached to the microslide using small columns of nail polish (Makarevich et al., 2005). Embryo diameter Embryo diameter including zona pellucida, was measured from the images on the screen of the monitor using scale bar micrometer, which was previously calibrated on a 40 x objective and 10 x eyepiece. The diameter of the embryos was the mean of two measurements made perpendicularly to each other (Makarevich et al., 2006). Statistical analysis The differences between groups were evaluated statistically by the unpaired Student´s t-test.

RESULTS AND DISCUSSION Significantly higher ICM cell number (p<0.05) was found out in the control group (44.18±2.50) compared to the experimental group (36.22±2.45) (Table 1). Higher total cell number was found in the control embryos. However, difference between control and experimental group was not statistically significant (p<0.05). Blastocysts obtained in vivo had higher diameter as well than in vitro cultured embryos, but significant difference was not confirmed. We also did not find significant difference in apoptotic cell number, however higher number of these cells was recorded in the experimental group. The proportion of the ICM cells to total cell counts was similar both in the control and the experimental group (28.48 vs. 28. 46 %), respectively (Table 1). In our experiments we investigated influence of culture conditions on the rabbit embryo quality. One of the most important features determining embryo quality is number of blastomeres in ICM (Iwasaki, 1990; Uhm, 2009). We found out that cultured rabbit embryos had significantly reduced ICM cell number (36.22±2.45) compared to the control group (44.18±2.50), but the proportion of ICM to total cell counts did not differ between these two groups. The same results have been reported by Macháty et al. (1998), who checked number of cells in porcine embryos and by Giles and Foote (1995) in rabbit blastocysts, where the ratio ICM/total number of cells was similar to our results (21 – 30 %). Olexíková et al., (2006) reported similar total cell number in rabbit blastocyst (124±3.24) analysed by DAPI after in vitro incubation for 100 h at 37.5°C. This was also confirmed in the other paper of Olexíková et al. (2010), where they found similar total cell number (121.1±4.41) in rabbit embryos at blastocyst stage derived from superovulated female at 19-20 hpc and cultured

Original paper under in vitro conditions (k-DMEM with 10 % FCS at 37°C, 5 % CO2) up to 100 h. Higher total cell number (up to 379.5±22.2) in rabbit blastocyst was reported by Tao and Niemann (1999). Saenz-de-Juano et al. (2010) found different rabbit embryo diameters depended on day of gravidity (4th, 5th and 6th day) in range from 244-375 µm up to 2255 – 2822 µm including mucosa layer. In our experiment we observed lower embryo diameters (133.95±8.95), however we evaluated them without mucosa layer which eventual diameter conspicuously enlarge. The occurrence of apoptosis in preimplantation embryos indicates suboptimal culture conditions or the effects of experimental treatments (Makarevich et al., 2005). This founding corresponds with our results, where the higher proportion of apoptotic cells was observed in in vitro cultured rabbit embryos compared to in vivo ones, although the significant difference was not confirmed. Fabian et al. (2007) reported that apoptosis in intact embryos does not occur earlier than at the 16-cell stage of embryos. In case of early rabbit blastocysts, the proportion of apoptotic cells was 1.38 %, but mouse embryos showed higher proportion of apoptotic cells (6.60 %) at this developmental stage. Makarevich et al., (2008) observed the influence of microinjection and vitrification itself on the presence of apoptotic cells in rabbit embryos. They recorded lower proportion of apoptotic cells (5.70 %) in the embryos subjected only to microinjection compared to embryos subjected to combination of microinjection and vitrification (7.54 %).

CONCLUSION In conclusion, our results demonstrate significant difference in ICM cell number between in vivo and in vitro cultured rabbit embryos at blastocyst stage. No significant difference in total cell number, apoptotic index and embryo diameter was found, although control group had higher total cell number and embryo diameter and lower apoptotic cell number.

ACKNOWLEDGEMENT This work was supported by the Slovak Research and Development Agency under the contract No. APVV -0556-011 and the Operational Programme Research and Development funded from European Regional Development Fund (LAGEZ 26220120051).

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Original paper REFERENCES CHRENEK, P. – VAŠÍČEK, D. – MAKAREVICH, A. – UHRIN, P. – PETROVIČOVÁ, 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. – MAKAREVICH, A. V. 2005. Production of rabbit chimeric embryos by aggregation of zonafree nuclear transfer blastomeres. Zygote, vol. 13, 2005, p. 39-44. CHRENEK, P. – MAKAREVICH, A. V. – BAUER, M. – JURCIK, R. 2008. Development rate and allocation of transgenic cells in rabbit chimeric embryos. Zygote, vol. 16, 2008, p. 87-91. FABIAN, D. – MAKAREVICH, A. V. – CHRENEK, P. – BUKOVSKÁ, A. – KOPPEL, J. 2007. Chronological appearance of spontaneous and induced apoptosis during preimplantation development of rabbit and mouse embryos. Theriogenology, vol. 68 (9), 2007, p. 1271-1281. FOULADI-NASHTA, A. A. – ALBERIO, R. – KAFI, M. – NICHOLAS, B. – CAMPBELL, K. H. S. – WEBB, R. 2005. Differential staining combined with TUNEL labeling to detect apoptosis in preimplantation bovine embryos. Reprod. Biomed. Online, vol. 10, 2005, p. 497-502. GILES, J. R. – FOOTE, R. H. 1995. Rabbit Blastocyst: Allocation of Cells to the Inner Cell Mass and Trophectoderm. Mol. Reprod. Dev., vol. 41, 1995, p. 204-211. IWASAKI, S. – YOSHIBA, N. – USHIJIMA, H. – WATANABE, S. – NAKAHARA, T. 1990. Morfology and proportion of inner cell mass of bovine blastocysts fertilized in in vitro and in vivo. J. Reprod. Fert., vol. 90, 1990, p. 279-284. JIANG, H. S. – WANG,W.L. – LU, K. H. – GORDON, I. – POLGE, C. 1992. Examination of cell numbers of blastocysts derived from IVM, IVF and IVC of bovine follicular oocytes. Theriogenology, vol. 37, 1992, 229 (Abstr.). MACHÁTY, Z. – DAY, B. N. – PRATHER, R. S. 1998. Development of early porcine embryos in vitro and in vivo. Biol. Reprod., vol. 59, 1998, p. 451-455. MAKAREVICH, A. V. – CHRENEK, P. – ZILKA, N. – PIVKO, J. – BULLA, J. 2005. Preimplantation development and viability of in vitro cultured rabbit embryos derived from in vivo fertilized gene – microinjected eggs: apoptosis and ultrastructure analyses. Zygote, vol. 13, 2005, p. 125-137. MAKAREVICH, A. V. – CHRENEK, P. – FĽAK, P. 2006. The influence of microinjection of foreign

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Slovak J. Anim. Sci., 45, 2012 (4): 123-126 gene into the pronucleus of fertilized egg on the preimplantation development, cell number and diameter of rabbit embryos. Asian-Aust. J. Anim. Sci., vol. 19 (1), 2006, p. 1-5. MAKAREVICH, A. V. – CHRENEK, P. – OLEXÍKOVÁ, L. – POPELKOVÁ, M. – TURANOVÁ, Z. – OSTRÓ, A. – PIVKO, J. 2008. Post-thaw survival, cell death and actin cytoskeleton in gene-microinjected rabbit embryos after vitrification. Theriogenology, vol. 70 (4), 2008, p. 675-681. MORI, M. – OTOI, T. – SUZUKI, T. 2002. Correlation between the cell number and diameter in bovine embryos produced in vitro. Reprod. Domest. Anim., vol. 37, 2002, p. 181-184. OLEXÍKOVÁ, L. – MAKAREVIČ, A. V. – CHRENEK, P. – KUBOVIČOVA, E. – PIVKO, J. 2006. Vplyv hypertermie v podmienkach in vitro na vitalitu preimplantačných embryí králikov. Acta fytotechnica et zootechnica - mimoriadne číslo. 2006, p. 195-197. OLEXÍKOVÁ, L. – Makarevich, A. V. – Pivko, J. – Chrenek, P. 2010. Antibody to Hsp70 alters response of rabbit preimnplantation embryos to hyperthermia in vitro. Anim. Reprod. Sci., vol. 119, 2010, p. 130-136. PAPAIANNOU, V. E. – EBERT, K. M. 1988. The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo nad in vitro. Development, vol. 102, 1988, p. 793-803. SAENZ-DE-JUANO, M. D. – Peñaranda, D. S. – Marco-Jiménez, F. – Llobat, L. – Vicente, J. S. 2011. Differential mRNA expression in rabbit in vivo pre-implantatory embryos. Reprod. Domest. Anim., vol. 46 (4), 2011, p. 567-572. TAO, T. – NIEMANN, H. 2000. Cellular characterization of blastocysts derived from 4- , 8- and 16-cell embryos and isolated blastomeres cultured in vitro. Human Reproduction, vol. 15 (4), 2000, p. 881-889. TURANOVÁ, Z. – KOPRDOVÁ, L. – BAUER, M. – OLEXÍKOVÁ, L. – MAKAREVICH, A. V. – OSTRÓ, A. – ZIVCAK, J. – CHRENEK, P. 2008. Quality of rabbit transgenic embryos. Slovak J. Anim. Sci., vol. 41 (3), 2008, p. 109-112. UHM, S. J. – GUPTA, M. K. – CHUNG, H. J. – KIM, J. H. – PARK,CH. – LEE, H. T. 2009. Relationship between developmental ability and cell number of day 2 porcine embryos produced by parthenogenesis and somatic cell nuclear transfer. Asian-Aust. J. Anim. Sci., vol. 22 (4), 2009, p. 483-491. VAN SOOM, A. – VANROOSE, G. – DE KRUIF, A. 2001. Blastocyst evaluation by means of differential staining: a practical approach. Reprod. Domest. Anim., vol. 36, 2001, p. 29-35.

Evaluation of rumen fermentation kinetics of some by-products using in situ and in vitro gas production technique

H. PAYA1, A. TAGHIZADEH1, S. LASHKARI2, S. SHIRMOHAMMADI1 Department of Animal Science, Faculty of Agriculture, University of Tabriz, Iran Department of Animal Science, Faculty of Agriculture, University of Kurdistan, Iran

1 2

ABSTRACT This study was carried out to determine the chemical composition and estimation of nutritive value of brewers’ grain, apple pomace, orange pulp and lemon pulp by-product using in vitro gas production technique. Test feeds had significant differences in chemical composition. Brewers’ grain had the highest (P<0.05) crud protein (19.5 % DM) and ether extract (7.8 % DM). Apple pomace contained the highest neutral detergent fibre (43.3 % DM), acid detergent fibre (32.3 % DM) and lowest ash (3 % DM). Cumulative gas production was recorded at 2, 4, 6, 8, 12, 16, 24, 36, 48 and 72 h of incubation and the equation Y = A (1-e-ct) was used to describe the kinetics of gas production. Potential gas production (A) and rates of gas production (c) differed (P<0.05) among feeds. Apple pomace showed higher potential gas production (A) (341 ml g-1 DM) and orange pulp had higher rate of gas production (c) (0.09 h-1) than the other feeds. Inversely, lemon pulp (220 ml g-1 DM) had lower potential gas production than the other test feeds. The metabolizable energy (ME) (MJ kg-1 DM) content of feeds was calculated using gas production data. According to gas production data, the ME values ranged from 7.66 in lemon pulp to 10.83 MJ kg-1 DM in apple pomace. For in situ technique duplicate dacron bags were incubated for 0, 2, 4, 8, 12, 24, 48 and 72 h in two wethers fitted with ruminal cannulas. The model y = a+b (1-e-ct) was used for determination of degradation characteristics. Orange pulp had higher soluble DM (a) (36.9 %), lemon pulp had higher insoluble potentially degradable DM (b) (62 %) and orange pulp had higher degradation rate (0.112 % h-1) than other feeds. It was concluded that regarding different chemical composition of test feeds, the in situ dry matter degradability, in vitro gas production and ME, SCFA and OMD of feeds showed different values. Key words: by-product; nutritive value; gas production; in situ; dry matter degradability

INTRODUCTION A major constraint to increasing livestock productivity in developing countries is the lack and fluctuating quantity and quality of the year-round supply of conventional feeds. These countries experience serious shortages in animal feeds of the conventional type. In order to meet the projected high demand of livestock products and to fulfil the future hopes of feeding the millions and safeguarding their food security, the better utilization of non-conventional feed resources which do not compete with human food is imperative. There is also

*Correspondence: E-mail: hamid.paya@tabrizu.ac.ir Hamid Paya, Department of Animal Science, Faculty of Agriculture, University of Tabriz, Iran Tel.: 0098 914 309 3006

a need to identify and introduce new and lesser known food and feed crops. Most by-product feedstuffs (BPF) result from the processing of commercial crops, the food processing industry and the fibre industry. Tobias Marino et al. (2010) studied the potential of several vegetables and fruit wastes, that had expired the date of display in supermarket shelves, as a ruminant feed source by in vitro gas production technique. They reported that some vegetables and fruits have potential as a ruminant feed. However, low dry matter content of these feeds can interfere with the viability of their transport and utilization.

Received: July 5, 2012 Accepted: November 20, 2012

127

Original paper However, little is known about their fermentation pattern in the rumen and a better understanding of their digestion and products of fermentation is necessary in order to properly balance their introduction into the diets (Durand et al., 1998; Sutton, 1986) and the knowledge about their potential feeding value is insufficient. Considerable amount of grains are used in the brewery and distillery industry. The main by-products are distillers‘ grains and distillers‘ solubles. Approximately, 100 kg of grains provides ±33 kg distillers‘ dried grains with solubles or ±20 kg distillers‘ dried grains and ±32.5 kg condensed distillers‘ solubles with ±35 % DM (Mirzaei-Aghsaghali and Maheri-Sis, 2008). Apple pomace is the by-product of the production of cider and juice. It accounts for about 18.5 kg wet or 4.2 kg dried apple pomace per 100 kg apples. Dried apple pomace is a source of pectin (Boucque and Fiems, 1988). The extraction of the juice from citrus fruits provides citrus pulp as residue. Citrus pulp consists of 60-65 % peels, 30-35 % segment pulp and 0-10 % seeds. There is little information available on the nutritive value of Brewers’ Grain (BG), Apple Pomace (AP), Orange Pulp (OP) and Lemon Pulp (LP). The present study was, therefore, carried out to determine the chemical composition, digestibility and degradability of BG, AP, OP and LP. There are several methods to evaluate feedstuffs. Determining the digestibility of feeds in vivo is laborious, expensive, requires large quantities of feed, and is largely unsuitable for single feedstuffs thereby making it unsuitable for routine feed evaluation. In vitro methods provide less expensive and more rapid alternatives (Getachew et al., 2004). Digestibility may be directly determined in vivo or estimated by using in vitro procedures, which are cheaper and more convenient (Aregheore, 2000). There are a number of in vitro techniques available to evaluate the nutritive value of feeds at relatively low cost such as in vitro gas production technique. The in situ nylon-bag technique is widely used to characterize the disappearance of feeds from the rumen (Woods et al., 2002). Nylon-bag technique provides a useful means to estimate rates of disappearance and potential degradability of feedstuffs and feed constituents (Getachew et al., 1998). Kamalak et al. (2005) compared in vitro gas production technique with in situ nylon bag technique to estimate dry matter degradation and reported that the in vitro gas production technique has good potential to predict in situ DM disappearance and some DM degradation parameters. In this study, in situ and in vitro gas production techniques were used to describe nutritive value of byproducts of the food industry for ruminants.

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Slovak J. Anim. Sci., 45, 2012 (4): 127-133 Material and Methods Experimental feeds The samples of BG, AP, OP and LP were collected from food industrial company in north-western Iran. BG and AP samples were moist and OP and LP samples were dry when collected. Samples of all test feeds for the in situ and gas production technique were milled through a 2.0 mm sieve and for chemical analyses they were milled through a 1.0 mm sieve. Moist samples were dried at 60°C before milling. Chemical analysis Samples of feeds and faeces were dried in an oven at 105°C for 24 h and the DM content calculated. Ground samples were analyzed for ash (AOAC, 2005). Determinations of N were conducted using the Kjeldahl method in an automated Kjelfoss apparatus (Foss Electric, Copenhagen, Denmark). Neutral-detergent fibre (NDF) and acid detergent fibre (ADF) were determined by the detergent procedures of Van Soest et al. (1991). Ether extract (EE) was determined by extracting the sample with ether (AOAC, 2005). In situ ruminal procedure Two wethers fitted with rumen cannula were used to measure rumen degradability of feeds. The wethers were fed a diet composed of 385g.day-1 lucerne hay, 280g. day-1 barley grains, 35 g.day-1 wheat bran and 1.5 g.day-1 limestone at maintenance on DM basis (NRC, 1985). The wethers were kept in individual tie-stalls with individual feed bins in an animal house and had continuous access to water. Diets were given as total mixed ration with fresh feed offered twice a day (08:30 and 15:30 h). The nylon bag technique (Orskov and McDonald, 1979) was used to measure the DM degradation of feeds in the rumen. Nylon bags (4 cm×8 cm polyester bag; pore size 45-50 µm) containing 3 g of feed ground through a 2 mm screen were incubated in the rumen for 2, 4, 8, 12, 24, 48 and 72 h for feeds, immediately after the morning feeding. As a whole, there were four replicates for each feed sample and for each incubation time (2 wethers × 2 bags). Immediately after removal from the rumen, the bags were washed in cold water and frozen at −18°C. At the end of the collections, they were unfrozen and washed together with the zero time bags (not incubated in the rumen) for 20 min and then dried at 80°C for 24 h. The residues were weighed and submitted for analysis. In vitro gas production Samples (300 mg) were weighed into 100 ml serum vial. Mc Dougall (1948) buffer solution was prepared and placed in a water bath at 39°C. Rumen liquor samples were obtained from the two wethers used

Slovak J. Anim. Sci., 45, 2012 (4): 127-133

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for in situ technique. Rumen fluid was collected after the morning feeding. Rumen fluid was pumped with a manually operated vacuum pump and transferred into pre-warmed thermos flask, combined, filtered through four layers of cheesecloth and flushed with CO2. Each feed sample was incubated in five replicates with 20 ml of rumen liquor and buffer solution (1:2). Five vials containing only the rumen fluid/buffer solution and no feed sample was included with each test and the mean gas production value of these vials was termed the blank value. The vials were sealed immediately after loading and were affixed to a rotary shaker platform (lab-line instruments Inc Melors dark, USA) set at 120 rpm housed in an incubator. Gas production was measured in each vial after 2, 4, 8, 12, 16, 24, 36, 48 and 72 h of incubation using a water displacement apparatus (Fedorak and Hrudey, 1983). Calculations and statistical analysis In situ dry matter (DM), rapidly degradable fraction (a), potentially degradable fraction (b), and rate of degradation of fraction b (c), were calculated according the model of Ørskov and McDonald (1979) as y = a+b (1-e-ct) that y is the actual degradation of DM after t, a is the intercept of the degradation curve at time zero, b is the potential degradability of the component of the insoluble but degradable DM, which will in time be degraded, c represents the constant of degradation rate of b at time t, t is incubation time. Rate and extent of gas production was determined for each feed by fitting gas production data to the one component McDonald model: Y = A (1- e-ct), where y is the volume of gas produced at time t, A the potential gas production (ml g-1 DM), and c the fractional rate of gas production. Parameters A and c were estimated by an iterative least square method using a non-linear regression procedure of the statistical analysis systems (SAS, 1999).

The metabolizable energy (MJ.kg-1 DM) content of feeds was calculated using equation of Getachew et al. (2002) as:

ME (MJ.kg-1DM) = 1.06 + 0.157GP+ 0.084CP+ 0.22CF − 0.081CA

The short chain fatty acid (SCFA) and organic matter digestibility (OMD) for feeds were calculated using equations of Menke et al. (1979) as: SCFA mmol. 200 mg-1 DM = 0.0222 GP - 0.00425 OMD (%) = 14.88 + 0.889 GP + 0.45 CP + CA Where, GP is 24 h net gas production (ml/200 mg DM); CP, CF and CA are crude protein, crude fat and crude ash (% DM), respectively. Data on in situ DM degradability and gas production parameters were subjected to one-way analysis of variance using the analysis of variation model (ANOVA) of SAS (1999). Multiple comparison tests used Duncan’s multiple-range test (Snedecor and Cochran, 1989).

RESULTS AND DISCUSSION Chemical composition The chemical composition of test feeds is presented in the Table 1. The CP content of feeds ranged from 66g.kg-1 in LP to 195g.kg-1 in BG. The NDF content of feeds ranged from 124g.kg-1 in OP to 433g.kg-1 in AP. Apple pomace contained substantially higher OM level than the other feeds. Variation in test feeds chemical composition has been observed compared to other studies with test feeds (Brabander, 1999, Kafilzadeh, 2008 and Bampidis and Robinson, 2006). The differences among chemical composition test feeds can be due to variation in varieties, cultivating and environmental conditions of feeds that are used in food industry. The feeds used in the current study were by-product feeds, which are created as a result

Table 1: Chemical composition of the test feeds (g.kg-1). †

Feedstuffs

Variable

Brewers’ grain

Apple pomace

Orange pulp

Lemon pulp

368

311

849

872

195

NDF

219

433

224

209

ADF

129

323

153

164

Hemicellulose

110

Ash

† Three samples were analyzed for each feed

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Slovak J. Anim. Sci., 45, 2012 (4): 127-133

of processing of fruits for human foods, and therefore their composition varies depending on the composition of original plant material, method of processing, and type of components extracted or removed (Getachew et al., 2004). In vitro gas production There was a difference (P<0.05) in gas production among feeds (Table 2). Potential gas production (A) and rates of gas production (c) differed (P<0.01) among feeds. The pattern of fermentation of test feeds was significantly different, particularly at first times of incubation (Figure 1). Apple pomace fermented faster and lemon pulp fermented slower than other test feeds. The strong correlation between extent of gas production and chemical composition, and the poor correlation between rate of gas production and chemical composition, is consistent with Nsahlai et al. (1994). Low gas yield for lemon pulp in incubation times compared to

the other test feeds resulted due to high content of ash and low content of crude protein. Low degradable CP was limiting microbial activity and low carbohydrate fractions degraded according to their potential. The high positive correlation among gas production, dry matter and organic matter digestibility was reported (Datt and Singh, 1995). Al-Masri (2003) reported a very highly significant (P<0.0001) relationship between gas production and the true and apparent fermented organic matter. The high level of orange pulp gas yield in initial incubation times can be assumed that orange pulp had high level of soluble carbohydrate. Taghizdeh et al. (2008) reported that amount soluble carbohydrate can alter gas yield in initial incubation time. Tamminga (1994) reported that particle size is one of the important factors that can alter gas yield and low particle size increase gas yield because microorganisms can be better attached and degradation rate can be increased. Citrus pulps have an especial physical structure

Table 2: In vitro gas production characteristics of feed samples incubated in buffered rumen fluid

Gas production (ml. g-1 DM)

Feeds

2 h

4 h

6 h

100

40b

85a

39b

SEM

(n=5)

a, b, c, d Means within a column with different superscripts differ (P<0.05). c: fractional rate of gas production (h-1); A: potential gas production (ml.g-1 DM). BG = brewersâ&#x20AC;&#x2122; grain, AP = apple pomace, OP = orange pulp and LP = lemon pulp.

16 h

24 h

36 h

48 h

132

169

214

244

273

292 313

308

122a

158a

209a

236a

279a

302a

315a 364a

341.3a

0.07b

106

130

175

196

209

218

245 256

244.6

0.09a

66b

81c

98c

141c

166d

183d

202c

219d 220d

220.9d

0.07b

4.08

5.07

6.47

5.04

5.00

5.57

5.55

4.489

0.0001

1.98 3.84

72 h

12 h

8 h

Gas production constants

4.84

0.06b

Table 3: The in situ disappearance of DM (%) and DM degradation characteristics of test feeds in the rumen

DM degradability (% DM)

Degradation characteristics

Feeds

0 h

2 h

4 h

8 h

12 h

24 h

48 h

72 h

a (%)

b (%) C (%.h-1)

21.7d

27.9d

32.2c

41.5d

51.4d

58.0d

66.0c

69.8c

21.6d

47.0c

0.070b

26.2c

30.4c

37.2b

47.1c

58.3c

66.7c

75.9b

77.5b

24.8c

52.7b

0.072b

40.1

42.7

54.6

68.8

75.9

85.4

87.4

87.6

36.9

51.3

0.112a

34.1b

37.7b

38.4b

59.8b

64.0b

87.9a

88.5a

89.8a

29.6b

62.0a

0.074b

SEM

(n=4)

0.473

0.576

0.700

0.722

0.621

0.552

0.613

0.667

0.541

0.834

0.0033

BG = Brewersâ&#x20AC;&#x2122; grain, AP = Apple Pomace, OP = Orange pulp and LP = Lemon Pulp. a, b, c, d Means within a column with different subscripts differ (P<0.05).

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Fig. 1: Pattern of in vitro gas production on incubation of test feeds in buffered rumen fluid

Fig. 2: Pattern of in situ degradability on incubation of test feeds in rumen

and are converted to powder when they are milled. In this study, citrus pulp had high gas yield in initial time and that’s because of using low particle size (powder form) of the feeds during incubation. Besharti et al. (2008) reported the value of 264 ml.g-1DM for gas yield after 24h incubation for apple pomace and in this study the value was 279 ml.g-1DM. This difference can result from variation in apple variety and apple pomace which can be a byproduct of juice or puree making industry which they have different chemical and nutrient composition. Kafilzade et al. (2008) reported that apple pomace from puree making, had a higher soluble carbohydrate than apple pomace from juice.

sufficient for test feeds to be degraded. A large range of dry matter degradation characteristics was obtained: the ‘a’, ‘b’ and ‘c’ values ranged from 21.6 to 36.9 % (for BG and OP), 47 to 62 % (for BG and LP) and 0.070 to 0.112 %h-1 (for BG and OP), respectively. The relationship between the degradability parameters a, b and c and the chemical composition of 60 test feeds was reported by Woods et al. (2003). They reported that the slowly fermented structural carbohydrates are thought to play a dominant role in the degradation characteristics in the rumen. The high level of orange pulp and lemon pulp DM degradability in several incubation times can be assumed that small particle size of milled orange pulp and lemon pulp (because of especial physical structure) was not limiting microbial activity thus allowing the orange pulp and lemon pulp be degraded according to their potential. In comparison to Pereira et al. (1998), DM degradation characteristics of brewers’ grain - the ‘a’ and ‘c’ values were similar and ‘b’ value was different in this study. Where different degradability values are observed between this study and that of Pereira et al. (1998), it is possibly because of differences in nutrient composition, pore size of nylon bag and milling screen size.

In situ method Dry matter losses from the nylon bags incubated in the rumen and in situ DM degradability characteristics are presented in Table 3. There were differences (P<0.05) among test feeds in dry matter degradability after several incubation times. Total washing losses of DM (zero time bags) represented 21.7 to 40.1 % of DM in brewers’ grain and orange pulp, respectively. Dry matter disappearance from nylon bags incubated in the rumen increased with increasing incubation time. The 72 h incubation time was

Table 4: Evaluated SCFA, ME and OMD by in vitro gas production results

Feedstuffs

According to in vitro gas production data

SCFA mmol. 200mg-1 DM

ME MJ.kg-1 DM

OMD %

Brewers’ grain

0.906

10.46

65.1

Apple pomace

1.236

10.83

70.7

Orange pulp

0.924

8.30

60.3

Lemon pulp

0.809

7.66

55.5

SCFA = short chain fatty acid, ME = metabolizable energy and OMD = organic matter digestibility

131

Original paper Metabolizable Energy (ME) and Short Chain Fatty Acids (SCFA) According to studies Menke et al. (1979) and Getachew et al. (2002), SCFA, ME and OMD could be evaluated by 24 h in vitro gas production data. These results are shown in Table 4. Low content of lemon pulp‘s SCFA, metabolizable energy and organic matter digestibility can result from its low rate of gas production, extent of gas production at 24 h and its nutrient composition.

CONCLUSION In the present study, results indicated that byproducts can be used as replacement feedstuffs in diets for ruminants. In an overall conclusion the nutritive value of apple pomace were better than other by-product feeds. However, all by-product feeds that were used in this study can be used economically as potential fibrous and energy sources in ruminant nutrition. As a whole, the wide variation in chemical composition of feedstuffs, gas production, rumen dry matter degradability, ME, SCFA and OMD offer users flexibility in formulating rations according to the productive performance of target animals.

ACKNOWLEDGMENT This work was supported by the Tabriz University and funded by the student scientific association central office, Ministry of Science, Research and Technology, I. R. Iran. Authors specially thank Dr Afshin Mottagi and Yusef Daneshi for their attention.

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Slovak J. Anim. Sci., 45, 2012 (4): 127-133 MENKE, K. H. – RABB, L. – SALEWESKI, A. – STEINGASS, H. – FRITZ, D. – SCHNIDER, W. 1979. The estimation of the digestibility and metabolizable energy content of ruminant feed stuffs from the gas production when they are incubated with rumen liquor in vitro. Journal of Agriculture Science, vol. 93, 1979, p. 217-222. MIRZAEI-AGHSAGHALI, A. – MAHERI-SIS, N. 2008. Nutritive value of some agro-industrial by-products for ruminants-A review. World J. of Zoology, vol. 3, 2008, p. 40-46. National Research Council. 1985. Nutrient Requirements of sheep. The National Academies Press, Washington DC, USA. ISBN 0-309-03596-1 NSAHLAI, I. V. – SIAW, D. E. K. A. – OSUJI, P. O. 1994. The relationship between gas production and chemical composition of 23 browses of genus Sesbania. J. of Sci. of Food and Agriculture, vol. 65, 1994, p. 13-20. ORSKOV, E. R. – McDONALD, Y. 1979. The estimation of protein degradability in the rumen from determining the digestibility of feeds in the rumen. The Journal of Agricult. Sci., vol. 92, 1979, p. 499-503. PEREIRA, J. C. – CARRO, M. D. – GONZALEZ, J. – ALVIR, R. – RRODIGUEZ, C. A. 1998. Rumen degradability and intestinal digestibility of brewers grains as affected by origin and heat treatment and of barley rootlets. Anim. Feed Sci. and Technology, vol. 74, 1998, p. 107-121. SNEDECOR, G. W. – COCHRAN W. G. 1989. Statistical Methods. Iowa State University Press, USA. Static Analysis Software. 1999. Static Analysis Software, Version release 8/0, SAS Institute Inc., Cary, NC, USA. SUTTON, J. D. 1986. Rumen fermentation and gastro-intestinal absorption: carbohydrates.

Original paper

p. 21-38. In: NEIMANN-SORENSEN A. (Ed.): New Developments and Future Perspectives in Research on Rumen Function. Forsogsanloeg Foulum, Denmark. ISBN 92-825-3467-7 TAGHIZADEH, A. – JANMOHAMADI, H. – MOGHADAM, G. A. – SHODJA, J. 2008. In vitro gas production profiles in some concentrate ingredients. J. of Anim. and Vet. Advances, vol. 7, 2008, p. 137-139. TAMMINGA, S. 1994. Feed processing as a means to improve feed utilisation by ruminants. Wageningen Institute of Animal Sciences, Wageningen Agricultural University, Netherlands. ISBN 90-5808-817-0 Tobias Marino, C. – Hector, B. – Mazza Rodrigues P. H. – Oliveira Borgatti, L. M. – Marques Meyer, P. – Alves da Silva, E. J. – Ørskov, E. R. 2010. Characterization of vegetables and fruits potential as ruminant feed by in vitro gas production technique. Livestock Research for Rural Development, vol. 22 (9), 2010. VAN SOEST, P. J. – ROBERTSON, J. B. – LEWIS, B. A. 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. of Dairy Sci., vol. 74, 1991, p. 3583–3597. WOODS, V. B. – O‘MARA, F. P. – MOLONEY, A. P. 2002. The in situ ruminal degradability of concentrate feedstuffs in steers as affected by level of feed composition and ratio of grass silage to concentrate. Anim. Feed Sci. and Technology, vol. 100, 2002, p. 15-30. WOODS, V. B. – O‘MARA, F. P. – MOLONEY, A. P. 2003. The nutritive value of concentrate feedstuffs for ruminant animals. Part I: in situ ruminal degradability of Dry matter and Organic matter. Anim. Feed Sci. and Technology, vol. 110, 2003, p. 111-130.

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QUALITY IMPROVEMENT OF SLOVAK EWE MILK BASED ON THE CONTENT OF HEALTH AFFECTING FATTY ACID COMPOUNDS

L. SOJÁK1, R. KUBINEC1, J. BLAŠKO1*, R. ČABALA3, W. LORENZ4, D. APOLEN2, M. MARGETIN2 Comenius University in Bratislava, Slovak Republic Animal Production Research Centre Nitra, Slovak Republic 3 Charles University in Prague, Czech Republic 4 Martin Luther University of Halle-Wittenberg, Halle, Germany 1 2

ABSTRACT The content of 70 C4-C24 fatty acids (FA) in bulk milk samples of dairy ewes at 4 farms during pasture season and at one farm during winter season was determined by gas chromatography. The FA content in milk fat of grazed ewes was higher up to 4-fold for rumenic acid (CLA), 3-fold for trans-vaccenic acid (TVA) and 2-fold for α-linolenic acid (ALA) compared to that during winter diet. The content of CLA (3.5 %) and TVA (7.9 %) in milk fat of grazed ewes from Tajov farm was higher than that in milk of pasture grazed ewes or cows published previously. Nevertheless, the farm at Trenčianska Teplá because of lower altitude and corresponding climatic conditions in summer showed temporal variation up to 50 % lower content of CLA and TVA. The variations in CLA content during pasture season are primarily related to the seasonal changes of ALA content in pasture plants. These findings suggest that further development of ewes´ milk production should be situated in the northern part of Slovakia at higher altitudes thus providing better quality pastures and more consistent FA composition of ewe milk products with a high content of health affecting FA compounds. Ewe individuality is another important factor significantly affecting the FA milk fat content and also milk yield whereas the ewes´ breed and parity had only little effect. The effect of individuality, breeds and parity of grazed ewes based on FA content was investigated in herd of 148 Tsigai, 124 Improved Valachian and 56 Lacaune ewes grazing pasture and milk samples of individual ewes were taken on the same day. The CLA milk fat content varied up to 5-fold and milk yield up to 12-fold among individual ewes. An inverse relation between the milk yield and CLA content was observed which was not significant though. The ewes with a higher CLA milk fat content and a corresponding higher milk yield were considered in ewe selection for improving milk quality based on the content of health affecting FA compounds. Upon eliminating the data for 25 % ewes with a lower CLA milk fat content and lower milk yield the average CLA milk fat content increased by 10 % while keeping the milk yield. Key words: ewes´ milk; fatty acids; CLA content; effect of diet; effect of individual ewes

INTRODUCTION The fatty acid (FA) profile of raw milk influences the nutritional and health characteristics of milk products. Milk FA have various effects on human health. The quality of dietary lipids could be an important modulator of the morbidity and mortality associated with obesity, arteriosclerosis, diabetes, hypertension, hyperlipidemia and cancer (Nagao and Yanagita, 2005). Conjugated FA have attracted considerable attention

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because of their potentially beneficial biological effects of attenuating these diseases. An increasing interest in enhancing the conjugated linoleic acid (CLA) content in food products arises from its potential anticarcinogenic, anti-atherogenic, anti-diabetic, antiobesity, and immunomodulatory functions observed in animal models. Cis-9,trans-11 18:2 is the major CLA isomer which represents 75 to 90 % of the total CLA in milk fat. This CLA isomer is an intermediate in the biohydrogenation of linoleic acid (LA), the main source

*Correspondence: E-mail: blasko@fns.uniba.sk Jaroslav Blaško, Institute of Chemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská dolina CH2, 842 151 Bratislava, Slovak Republic Tel.: +421 2 60296 330 Fax: +421 2602 96 337

Received: September 18, 2012 Accepted: November 26, 2012

Slovak J. Anim. Sci., 45, 2012 (4): 134-143 in milk fat is from endogenous synthesis in the mammary gland. An intermediate in the rumen biohydrogenation of linoleic and linolenic acid (ALA) is trans-vaccenic acid, trans-1118:1 (TVA), which is converted to cis-9,trans-11 18:2 by enzyme Δ9-desaturase in the mammary gland. Polyunsatured fatty acids (PUFA), such as LA, 18:2, n-6, ALA, 18:3, n-3 and arachidonic acid (AA, 20:4, n-6) being essential FA are very important for maintaining biological functions in mammalians. The intake of n-3 highly unsaturated FA, such as eicosapentaenoic acid (EPA, 20:5, n-3) and docosahexaenoic acid (DHA, 22:6, n-3), correlates with a reduced risk of cancer and cardiovascular disease. On the other hand, some saturated FA, such as myristic acid (MA, 14:0) and palmitic acid (PA, 16:0) and some unsaturated trans-FA show adverse effects on health maintenance and disease prevention. There are several endogenous and exogenous factors affecting the ewes’ milk FA profile, such as diet (Addis et al., 2005; Cabiddu et al., 2005), season (Nudda et al., 2005; Meľuchová et al., 2008), climate (Meľuchová et al., 2009), and physiological factors such as individuality of animals (Tsiplakou et al., 2006; Soják et al., 2012), breed (Signorelli et al., 2008; Soják et al., 2012), lactation stage and parity (Tsiplakou et al., 2006; Soják et al., 2012). Previous studies were focused mainly on most significant dietary sources of FA variation, whereas the effect of physiological and genetic factors, particularly the effect of individuality of ewes on milk FA profile was investigated to a lesser degree. Previous studies suggested that not only diet but also selection of individual ewes may serve as an important tool to improve the nutritional health quality of milk fat. In the light of recommendation aimed at lower intact of milk products (German and Dillard, 2004), contemporary research is focused on producing the milk with higher FA content that may have beneficial effects on human health. Carta et al. (2008) suggested that for the FA profile of ewes´ milk both classical quantitative genetic approach and genomic information based selection approaches will be realistic options in the future. The most important product of ewe farming in Slovakia is a bryndza cheese with yearly production of about 5 x 106 kg. The EC regulation No. 676/2008 of July 16, 2008 registered the name Slovenská bryndza in the Register of protected designations of origin and protected geographical indications. Nevertheless, the detailed composition of individual FA of bryndza cheese has not been established. The FA profile of bryndza cheese corresponds to those in the milk fat the product is being produced from. Therefore, the content of individual 70 C4-C24 FA in milk fat of ewes fed with total mixed ration (TMR) during winter as well as that of ewes grazing on natural pasture (experimental flock of 350 ewes) was measured as bulk milk samples. Effect of individuality of ewes as well as breed, parity and

Original paper milk yield on the milk FA profile was investigated on the similar experimental flock belonging to three breedsTsigai, Improved Valachian, and Lacaune. In this case, the milk of grazed individual ewes was sampled on the same day due to the effects of differences in pasture management, botanical composition of pasture and vegetative stages of plants. The aim of this study was to determine the quality of Slovak ewe milk based on individual FA composition and to investigate options for improvement of ewe milk FA profile of those grazing on natural pasture as well as fed with winter diet.

MATERIAL AND METHODS Milk samples collected from ewes bred at Trenčianska Teplá farm were analyzed. This experimental farm kept 350 dairy ewes belonging to three breeds Tsigai, Improved Valachian, and Lacaune with parity 1-8. In winter season from mid-February to mid-April, the ewe basic diet consisted of corn silage (2.5 kg), meadow and Lucerne hay (0.7 kg and 0.3 kg), commercial concentrate feed mixture (0.8 kg), and mineral supplement (0.02 kg). In the grazing season from mid-April to mid-September, the ewes grazed natural pasture and also received a concentrated feed at a dose of 0.2 kg/day during machine milking. The FA composition of bulk ewe milk from the daily milkings was analyzed at least once in a month, and more frequently in milk of ewes fed TMR and at the beginning of the pasture season (April-June during 2007-2009). On days of milk sampling during pasture season, botanical families and main plant species of pasture samples on the content of individual FA were also analyzed (Meľuchová et al., 2008). For evaluation of the effect of ewe individuality on FA profile, individual milk samples from 328 ewes, 148 Tsigai, 124 Improved Valachian, and 56 Lacaune, grazing pasture were collected on the same day on morning milking in the year 2009. For completion of data evaluation, the bulk milk products of ewes grazed on pasture from Ružomberok, Liptovská Anna, and Tajov farms breeding 300-800 ewes were analyzed, too. The lipids from milk samples and dried pasture plant samples were extracted using chloroform-methanol mixture (2:1). The extracts were derivatized by sodium methanolate in methanol and analyzed as methyl esters of fatty acids (FAME). For analysis of FAME from bulk milk samples capillary gas chromatography (GC) with flame ionization detector in capillary column 100 m x 0.25 mm i.d. x 0.25 µm film thickness of CPSil 88 (Varian, Palo Alto, CA, USA) as stationary phase was used. Gas chromatographically unseparated CLA isomers, mainly triplet trans-7,cis-9 / cis-9,trans-11 / trans-8,cis-10 isomers of CLA, were resoluted by chemometric deconvolution (Blaško et al., 2009). For

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Original paper quick analysis of milk FA of individual ewes the capillary column of 60 m x 0.25 mm i.d. x 0.25 µm film thickness of DB-23 (J&W Scientific, Agilent Technologies, USA) as stationary phase was used. Individual FAME separation was performed on a gas chromatograph Agilent Technologies 6980N (Agilent, Waldbrown, Germany) and a 5973 Network mass-selective detector and at a programmed column temperature of 70-240°C. Separated FAMEs were identified by reference materials, published retention data and mass spectrometric measurements. The chromatograms were evaluated quantitatively using a method of internal normalization and published response factors of flame ionization detector for FAME (Ackman, 2002). The FAME composition of milk fat was expressed as grams of each individual FAME per 100 g of sum detected FAME. The average relative standard deviation of analyzed FAME with content > 0.5 g 100 g-1 was 1.1 % for whole analytical procedure and 5 replicate milk samples. The contents of individual FA samples in bulk milk were analyzed using a one-way ANOVA statistical package. The data for individual animals were statistically evaluated using a general linear model procedure with nominal variables of breed and parity and continuous variable of milk yield. Significant differences were considered at the level P < 0.05.

RESULTS AND DISCUSSION The results of GC analysis of individual FA content in bulk milk samples of ewes fed with winter diet (TMR) and those grazed on natural pasture as well as that of pasture plant samples are presented in Table 1. The variations in CLA milk fat content during continuous transition from a period of TMR diet, a period of gradually increased content ratio of pasture /TMR, and a final period of pasture feeding are presented in Fig. 1. Cis-9,trans-11 CLA isomer and trans-vaccenic acid (TVA) showed more than 5-fold changes in the individual FA milk fat content among ewes fed winter diet and pasture which was the most significant. Ewe feeding with TMR during the first 60 days after lambing, except for colostrum period, resulted almost in consistent individual milk FA content, e.g., CLA 0.65 g 100 g-1 in agreement with a quasi-standardized TMR diet. Nevertheless, the ALA content of meadow hay as a part of TMR diet from various cuts of 32 Slovak producers was in broad content range from 14.6 to 53.5 % (g 100 g-1 FAME). Replacement of meadow hay as a part of TMR diet by that having a higher ALA content (50 g 100 g-1 versus 25 g 100 g-1) led to a two-fold increase of CLA (0.65 to 1.22 g 100 g-1 FAME) and TVA (1.44 to 2.58 g 100 g-1 FAME) content in milk of TMR-fed ewes.

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Slovak J. Anim. Sci., 45, 2012 (4): 134-143 Luna et al. (2008) reported a two-fold increase in CLA and TVA contents upon feeding ewes with a TMR diet enriched with flax seed and sunflower oil (CLA 0.47 vs. 0.85 g 100 g-1, and TVA 0.87 vs. 1.72 g 100 g-1 FAME). Generally, when compared with previously published data, the estimated CLA milk fat content in our ewes fed with a winter diet (0.65 g 100 g-1 FAME) was moderately higher due to more appropriate TMR composition.

Fig. 1: �� Temporal variations in CLA milk fat content of ewes during a period of TMR diet, a period of gradually increased content ratio of pasture / TMR diet, and a period of pasture feeding in Trenčianska Teplá farm.

Fig. 2: Temporal �� variations in the content of CLA in ewe milk fat and content of α-linolenic (ALA), linoleic (LA) and oleic (OA) acids in pasture samples during pasture season in Trenčianska Teplá.

Slovak J. Anim. Sci., 45, 2012 (4): 134-143

Original paper

Table 1: Milk fatty acid composition of ewes fed TMR, hay TMR aditived, grazing on pasture in TrenčianskaTeplá, Ružomberok, Tajov, Liptovská Anna and in pasture plants (g 100 g-1 FAME)

Trenč. Teplá

TMR

Ružomberok Tajov Lipt. Anna

Pasture (T. Teplá)

TMR+hay

May

July

May

September

May

C4:0 3.31 C6:0 2.43 C9:0 0.04 C8:0 2.23 C10:0 6.82 C10:1 0.16 C11:0 0.08 C12:0 4.01 C12:1 0.02 iso-C13:0 0.06 C13:0 0.09 iso-C14:0 0.11 C14:0 (MA) 9.21 C14:1 0.10 iso-C15:0 0.27 anteiso-C15:0 0.37 C15:0 0.83 C15:1 0.05 iso-C16:0 0.27 C16:0 (PA) 22.85 6-8t-C16:1 0.08 9-11t-C16:1 0.34 c9-C16:1 0.71 c11-C16:1 0.05 iso-C17:0 0.52 anteiso-C17:0 0.47 C17:0 0.66 C17:1 0.26 iso-C18:0 0.09 C18:0 12.01 t6-t8-C18:1 0.10 t9-C8:1 0.14 t10-C18:1 0.35 t11 C18:1 (TVA) 1.44 t12/c6-8-C18:1 0.24 c9/c10-C18:1 (OA) 21.43 c11/t15-C18:1 0.66 c12-C18:1 0.36 c13-C18:1 0.09 c14-C18:1+9t12t-C18:2 0.36 C18:2 0.35 c9t12-C18:2 0.11 t9tc12 + t11c15-C18:2 0.08 C18:2 n-6 (LA) 2.77 t11c15-C18:2 0.14 c9c15-C18:2 0.04 C18:3 n-6 0.02 C19:0+C18:2 0.13 C18:3+cyclo C18 0.14 C18:3 n-3 (ALA) 0.52

2.58 2.58 2.69 0.08 8.50 0.02 0.10 4.41 0.04 0.10 0.09 0.12 8.84 0.17 0.34 0.36 0.76 0.03 0.24 21.65 0.18 0.42 1.19 0.06 0.54 0.49 0.70 0.46 0.12 6.26 0.18 0.26 0.63 2.58 0.31 22.95 0.65 0.36 0.12 0.28 0.36 0.17 0.12 3.15 0.09 0.04 0.01 0.10 0.11 0.56

4.05 2.84 0.04 2.78 8.45 0.25 0.11 4.80 0.03 0.07 0.11 0.12 9.92 0.16 0.39 0.67 1.14 0.12 0.29 18.05 0.61 0.39 0.72 0.01 0.61 0.55 0.76 0.22 0.04 9.66 0.08 0.12 0.21 4.68 0.21 17.16 0.51 0.27 0.13 0.61 0.66 0.16 0.13 2.43 0.11 0.09 0.03 0.12 0.22 1.08

2.25 1.29 0.04 1.01 3.32 0.15 0.03 2.38 0.09 0.05 0.08 0.13 9.72 0.20 0.43 0.57 1.23 0.14 0.27 24.82 0.27 0.40 1.11 0.02 0.60 0.48 0.83 0.38 0.07 12.30 0.07 0.10 0.14 1.70 0.09 24.19 0.57 0.26 0.08 0.52 0.55 0.16 0.08 2.75 0.11 0.08 0.02 0.15 0.29 1.57

2.83 1.75 1.46 0.04 4.53 0.01 0.05 2.67 0.02 0.07 0.08 0.14 8.50 0.13 0.36 0.65 1.18 0.15 0.31 22.15 0.53 0.55 0.74 0.02 0.55 0.45 0.82 0.23 0.06 11.52 0.18 0.21 0.36 5.87 0.44 17.53 0.70 0.26 0.15 0.81 0.59 0.20 0.08 3.05 0.10 0.13 0.01 0.05 0.11 1.72

2.42 1.91 1.78 0.04 5.93 0.01 0.06 3.27 0.03 0.09 0.07 0.15 9.61 0.16 0.36 0.70 1.18 0.13 0.30 21.73 0.69 0.40 0.79 0.02 0.56 0.49 0.78 0.26 0.06 10.54 0.19 0.24 0.18 7.85 0.29 15.52 0.55 0.16 0.14 0.63 0.41 0.13 0.09 2.12 0.07 0.09 0.02 0.03 0.11 1.01

2.52 1.75 1.71 0.05 6.18 0.02 0.08 4.09 0.01 0.05 0.09 0.10 11.12 0.31 0.36 0.50 1.13 0.01 0.23 22.14 0.48 0.50 1.04 0.02 0.51 0.34 0.63 0.25 0.04 9.22 0.16 0.18 0.33 5.25 0.39 15.66 0.62 0.31 0.12 0.76 0.75 0.21 0.12 2.79 0.06 0.11 0.02 0.03 0.09 1.88

0.04 0.12 0.42 0.01 0.11 15.16 2.01 0.03 0.17 0.05 1.58 0.15 2.55 0.29 16.48 58.96

t7c9-C18:2

0.04

0.03

0.04

0.06

0.04

MA – myristic acid; PA – palmitic acid; TVA – trans-vaccenic acid; OA – oleic acid; LA – linoleic acid; ALA – α-linolenic acid

0.020

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Original paper

Slovak J. Anim. Sci., 45, 2012 (4): 134-143

Table 1: (continuous)

Trenč. Teplá

Ružomberok Tajov Lipt. Anna

Pasture (T. Teplá)

TMR

TMR+hay

May

July

May

September

May

c9t11-C18:2 (CLA) t8c10-C18:2 t9c11-C18:2 t10c12-C18:2 t11c13-C18:2 c9c11-C18:2 t12t14-C18:2 t11t13-C18:2 t7t9-t10t12-C18:2 C20:0 C20:1 C20:2 C21:0 C20:3 n-6 C20:4 n-6 (AA) C22:0 C20:5 n-3 (EPA) C23:0 C24:0 C22:5 n-3 (DPA) C22:6 n-3 (DHA

0.65 0.01 0.005 0.001 0.006 0.005 0.002 0.006 0.03 0.22 0.10 0.03 0.06 0.03 0.24 0.07 0.05 0.05 0.05 0.01 0.02

1.22 0.02 0.011 0.002 0.003 0.006 0.001 0.01 0.002 0.13 0.09 0.03 0.04 0.06 0.33 0.03 0.06 0.01 0.01 0.15 0.04

2.49 0.01 0.006 0.002 0.04 0.003 0.01 0.02 0.02 0.21 0.20 0.02 0.11 0.08 0.21 0.14 0.07 0.10 0.17 0.21 0.07

1.21 0.02 0.006 0.001 0.020 0.003 0.01 0.0 0.012 0.32 0.12 0.03 0.10 0.03 0.20 0.14 0.10 0.09 0.06 0.17 0.04

2.62 0.01 0.011 0.003 0.07 0.006 0.02 0.05 0.02 0.28 0.20 0.02 0.12 0.03 0.15 0.18 0.09 0.12 0.11 0.19 0.05

3.50 0.02 0.001 0.008 0.12 0.005 0.01 0.02 0.01 0.25 0.23 0.01 0.10 0.02 0.08 0.18 0.08 0.14 0.13 0.15 0.05

2.53 0.01 0.011 0.003 0.07 0.006 0.02 0.04 0.02 0.15 0.03 0.02 0.09 0.02 0.09 0.11 0.09 0.08 0.06 0.19 0.08

0.47 0.83 0.57 -

CLA – conjugated linoleic acid; AA – arachidonic acid; EPA – eicosapentaenoic acid; DPA – docosapentaenoic acid; DHA – docosahexaenoic acid.

Table 1 and Fig. 1 suggest a rise in CLA, TVA and ALA content in milk samples upon transition from a TMR diet to a pasture diet. CLA, TVA and ALA values have been increasing from a TMR period throughout a transition period to the beginning of pasture season (May) (P < 0.001). The CLA and TVA contents declined later (June-July) (P < 0.001), however, they rose again to the end of the pasture season from August to midSeptember (P < 0.001).The composition of FA in the milk of pasture-grazed ewes in September was similar to that at the beginning of pasture season in May. The content of CLA and TVA in milk of ewes grazing fresh pasture at the beginning or at the end of pasture season was up to 4-fold higher (P < 0.001) and that of ALA was double compared with those fed with standard TMR diet (P < 0.001). These findings were consistent with a higher ALA content in pasture (60 g 100 g-1 FAME) (Fig. 2). Lower CLA (up to 2-fold) and TVA contents in milk fat were noted during warm and dry summer (June-July) which were associated with the corresponding decrease in ALA content (60 vs. 40 g 100 g-1 FAME) in grazed pasture. About 2-fold increase in CLA and TVA contents (P < 0.001) in ewes’ milk fat noted between the end of July and mid-September was consistent with higher ALA content of pasture grass (60 vs. 40 g 100 g-1 FAME). Of note, Nudda et al. (2005) published a regular monotonic decrease of ewe milk CLA

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content during pasture season from 2.20 in March to 1.14 g 100 g-1 FAME in June. The contents of CLA (3.50 %) and TVA (7.85 %) in milk fat of ewes grazed on Tajov pasture were higher than those of cows and ewes grazing on pasture published so far (Collomb et al., 2006).

Fig. 3: Temporal �� variations in the content of CLA ewe milk fat during pasture season in ewe farm Trenčianska Teplá (250 m a.s.l.) and Ružomberok farm (800 m a.s.l.).

Slovak J. Anim. Sci., 45, 2012 (4): 134-143 Similar data on milk FA composition of ewes grazing pasture at Trenčianska Teplá in May and September were also found in other ewe farms in Ružomberok, Liptovská Anna and Tajov (Table 1). The comparison of seasonal variations of CLA milk fat content in Ružomberok and Trenčianska Teplá farms shown in Fig. 3 suggest that the effect of summer pasture period on CLA content from Ružomberok farm was not significant because of superior climate conditions associated with higher altitude of pastures (altitude: 800 m vs. 250 m a.s.l.). Ewes grazing pastures at higher altitudes (altitude: 800 m a.s.l.) in North Slovakia showed a more consistent composition of milk FA in the same pasture season. Bearing in mind global warming, the above findings suggest that further development of ewes’ milk production should be situated in a northern part of the country at higher altitudes thus providing more good-quality pasture and meadow hay for a dry winter diet as well as more consistent FA composition of ewe milk products with a higher content of health affecting FA compounds (Ostrovský et al., 2009). The observed CLA-ALA relationships suggest that the variation of CLA milk fat content during pasture

Original paper

Fig. 4: �� Temporal variations in the content of ALA and LA of pasture after reseeding the plants mixture compared with the control pasture in Trenčianska Teplá in 2008, 1 – oversowing pasture, 2- control pasture.

Fig. 5: Temporal �� variations of average daily temperature, average daily rainfall, and average CLA milk fat content during pasture season of years 2007, 2008 and 2009 in Trenčianska Teplá farm.

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Original paper season are primarily related to seasonal changes of ALA content in pasture plants. Preliminary pasture reseeding in Trenčianska Teplá farm was accomplished using a common oversowing mixture of seven plant species (32 kgha-1). Pasture reseeding resulted in a relative increase of 8 % in pasture ALA content in June-July, however, it decreased to a control level throughout pasture season (Meľuchová et al., 2009) (Fig. 4). In the next season we will investigate optimized oversowing the pasture with Lolium perenne and Trifolium repens showing quick connection to pasture, high cover and durability growth aimed at increasing the ALA content in pasture. The FA composition of ewe milk samples collected at 4 Slovak ewe farms (Trenčianska Teplá, Ružomberok, Liptovská Anna and Tajov) (altitude: 250 – 800 m a.s.l.) were compared with published data for cow milk collected at 4 Swiss alpine locations (altitude: 1275 – 2120 m a.s.l.) (Kraft et al., 2003). The milk and cheese contents of CLA, TVA and ALA were found to be rather similar despite significant altitude and botanical differences between these pastures. Consequently, altitude or botanical composition does not serve as a crucial factor for increasing the content of health promoting milk FA. A similar content of ALA in Slovak and Swiss pastures (about 60 %) provides comparably high CLA, TVA, and ALA contents in milk products of ewes and cows grazing on pasture. Trans-11, cis-13 CLA was the second most abundant CLA isomer in milk fat from cows grazing high-altitude Alpine pastures. Therefore, this isomer has been implicated as a useful indicator of milk products of Alpine origin. Nevertheless, we noted identical data for milk of ewes grazing on natural pastures with an altitude range of 250-800 m a.s.l. (Blaško et al., 2009; Soják et al., 2009). The content of trans-11,cis-13 CLA isomer in milk fat of ewes grazing on pasture is about 3-fold higher than that of trans-7,cis-9 CLA isomer being normally the second-most abundant CLA isomer in ruminant milk fat. Further improvement in FA milk fat profile can be achieved by selecting individuals from ewes’ flock with higher CLA milk fat content and higher milk yield. The current selection of dairy ewes does not consider the content of individual FA in milk fat. The study on the effects of inter-individual variation, breed, parity on milk FA profile and milk yield of individual ewes grazed on pasture of 328 ewes belonging to Tsigai (148), Improved Valachian (124), and Lacaune (56) breeds shows how these parameters might affect the composition of individual milk fat FA (Soják et al., 2012). To eliminate confounding dietary and seasonal effects on milk FA profile, the milk of ewes grazed on pasture was sampled on the same day (May 16, 2009). On the same day, the highest CLA milk fat content was found during 2007-2008 years (Fig. 5). In addition to diet, ewe individuality is another

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Slovak J. Anim. Sci., 45, 2012 (4): 134-143

Fig. 6:

�� Frequency distribution for CLA milk fat content of 328 individual ewes including 148 Tsigai, 124 Improved Valachian, and 56 Lacaune breeds of this flock May 16, 2009 in Trenčianska Teplá farm.

most important factor significantly affecting the milk FA profile. The CLA milk fat content varied up to 5fold among individual 328 ewes (from 0.5 to 2.5 g 100 g-1) feeding pasture on the same day (Fig. 6).The quasi Gaussian frequency distribution of individual ewes for CLA milk fat content of the whole flock, and separately for Tsiagi, Improved Valachian, and Lacaune breeds of this flock was found. In addition, the systematic differences in CLA milk fat content between ewe pairs of examined breeds were prevailingly maintained over 16-wk pasture season as well as in previous year (2008) (Fig. 7). Earlier, Peterson et al. (2002) and Kelsey et al. (2003) stated that cows maintained a quite consistent hierarchy in CLA milk fat content over time when cows fed with the same diet even when cows were switched between the diets that give substantial differences in CLA milk fat content. A profound impact of ewe individuality inhibits a less significant effect of breed and parity on milk FA composition. The study on effect of ewe breed on milk FA profile can be investigated for producing ewe cheese with different sensory properties (Signorelli et al., 2008). In our study, the average content of CLA and other FA of the whole flock and that of Tsigai, Improved Valachian and Lacaune breeds was similar and prevailingly not statistically different. Similarly, in the study of the effect of breed on milk FA content in Awassi, Lacaune, Friesland and Chios breeds, Tsiplakou et al. (2008) found no effect of ewes’ breed on milk FA profile. Animal parity is another physiological factor, which can affect FA profile in milk fat. Our results in

Slovak J. Anim. Sci., 45, 2012 (4): 134-143

Original paper

Fig. 7: Temporal �� variations in CLA milk fat content of pairs Lacaune, Tsigai and Improved Valachian ewes with most different CLA milk fat contents (from 15 investigated ewes) during continuous transition from dry winter to natural pasture diet.

ewes with parities 1-8 suggest a 60 % decrease in milk yield between ewes with 1-3 parities and those with 7 parities (Soják et al., 2012). A higher content of oleic acid by 13 % and lower content of myristic acid by 8 % was the most significant difference between primiparous and multiparous ewes in the entire flock and also in individual breeds. The content of CLA slightly changed with parity: it increased with parity 1-3 and then decreased with parities 6-7. The data document that ewe parities 1-3 have a little impact on FA milk fat profile. Ewe individuality markedly affected milk yield with variations up to 12-fold (100-1250 mL at the morning milking). Similarly, Haenlein (2001) found the differences in ewes´ milk yield about 10-fold. The average morning milk yield of investigated flock was 627 mL, decreased in the order of Lacaune (711 mL) < Improved Valachian (611 mL) ≈ Tsigai (609 mL). The quasi Gaussian frequency distribution of individual ewes as for CLA milk fat content of the whole flock, and separately for Tsigai, Improved Valachian and Lacaune breeds of this flock was observed also for milk yield (Fig. 8), as well as Δ9-desaturase index and atherogenicity index (Soják et al., 2012). Just like in the year 2008, the rank of individual ewes during pasture season based on milk yield was to a large extent maintained particularly for those with more different milk yield. The calculated linear regression equation between ewes’ milk yield and CLA milk fat content in individual ewes of investigated breeds suggested a non-significant inverse relationship between the CLA milk fat content and milk yield (Fig. 9). There seems to be only a trend

of decreasing CLA milk fat content with increasing milk yield. Therefore, the ewes with a higher CLA milk fat content and correspondingly higher milk yield should be considered in ewe selection. The annual replacement rate in an experimental ewe flock is 20-25 %. Upon eliminating the data for 25 % ewes with a lower CLA milk fat content and a lower milk yield, the average CLA milk fat content increased approximately by 10 % while keeping the

Fig. 8: Frequency �� distribution for milk yield (mL morning milking) of 328 individual ewes of flock and 148 Tsigai, 124 Improved Valachian, and 56 Lacaune breeds on May 16, 2009 in Trenčianska Teplá farm.

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Original paper

Slovak J. Anim. Sci., 45, 2012 (4): 134-143 content and a correspondingly higher milk yield should be considered in ewe selection for improving milk quality in experimental ewe flock. The ewe selection based on a single milk sample for each ewe on the same day from the experimental grazed flock was substantiated by prevailing maintenance of the systematic differences in CLA milk fat content as well as milk yield of individual ewes during pasture season as well as between pasture seasons.

ACKNOWLEDGEMENT This work was supported by the Slovak Research and Development Agency under contracts nos. APVV-0458-10 and APVV-0665-10.

REFERENCES Fig. 9: �� Relationship between CLA milk fat content and yield of morning milk of 328 individual ewes 148 Tsigai, 124 Improved Valachian, and 56 Lacaune breeds on May 16, 2009 in Trenčianska Teplá farm.

milk yield. In the case of higher starting CLA milk fat content this increase should be even higher. On the other hand, the average milk yield increased approximately by 15 % while keeping the average CLA milk fat content.

CONCLUSION The contents of CLA and TVA in milk fat of pasture-grazed ewes belong to the highest ever published for ruminant animals. They decreased in lowland pastures during summer months, and increased again at the end of pasture season up to the levels noted at the beginning of the season. The seasonal variations in milk FA contents were related to those of ALA content in pasture. The strategies aimed at increasing CLA milk fat content in ewes fed with a winter diet through replacing the meadow hay as a part of a winter diet with that having a higher ALA content, and in those grazing on pasture through reseeding pasture with plant species with a higher ALA content were investigated. In addition to diet, ewe individuality is another important factor significantly affecting the CLA milk fat content. Ewe individuality significantly affected milk yield too. Upon consideration of statistically not significant relationship between the CLA milk fat content and milk yield, the animals with a higher CLA milk fat

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ACKMAN, R. G. 2002. The gas chromatograph in practical analyses of common and uncommon fatty acids for the 21st century. Anal. Chim. Acta, vol. 465, 2002, p. 175-192. ADDIS, M. – CABBIDU, A. – PINNA, A. – DECANDIA, M. – PIREDDA, G. – PIRISI, A. – MOLLE, G. 2005. Milk and cheese fatty acid composition in sheep fed Mediterranean forages with reference to conjugated linoleic acid cis-9, trans-11. J. Dairy Sci., vol. 88, 2005, p. 3443-3454. BLAŠKO, J. – KUBINEC, R. – OSTROVSKÝ, I. – PAVLÍKOVÁ, E. – KRUPČÍK, J. – SOJÁK, L. 2009. Chemometric deconvolution of gas chromatographic unresolved conjugated linoleic acid isomers triplet in milk samples. J. Chromatogr. A, vol. 1216, 2009, p. 2757-2761. CABIDDU, A. – DECANDIA, M. – ADDIS, M. – PIREDDA, G. – PIRISI, A. – MOLLE, G. 2005. Managing Mediterranean pastures in order to enhance the level of beneficial fatty acids in sheep milk. Small Rumin. Res., vol. 59, 2005, p. 169-180. CARTA, A. – CASU, S. – USAI, M. G. – ADDIS, M. – FIORI, M. – FRAGHI, A. – MIARI, S. – MURA, L. – PIRREDA, G. – SCHIBLER, L. – SECHI, T. – ELSEN, J. M. – BARILLET, F. 2008. Investigating the genetic component of fatty acid content in sheep milk. Small Rumin. Res., vol. 79, 2008, p. 22-28. COLLOMB, M. – SCHMID, A. – SIEBER, R. – WECHSLER, D. – RYHÄNEN, E. L. 2006. Conjugated linoleic acids in milk fat: Variation and physiological effects. Int. Dairy J., vol. 16, 2006, p.1347-1361. GERMAN, J. B. – DILLARD, C. J. 2004. Saturated fats: what dietary intake? Am. J. Clin. Nutr., vol. 80, 2004, p. 550-559.

Slovak J. Anim. Sci., 45, 2012 (4): 134-143 KELSEY, J. A. – CORL, B. A. – COLLIER, R. J. – BAUMAN, D. E. 2003. The effect of breed, parity, and stage of lactation on conjugated linoleic acid (CLA) in milk fat from dairy cows. J. Dairy Sci., vol. 86, 2003, p. 2588-2597. KRAFT, J. – COLLOMB, M. – MÖCKEL, P. – SIEBER, R. – JAHREIS, G. 2003. Difference in CLA isomer distribution of cow’s milk lipids. Lipids, vol. 38, 2003, p. 657-664. LUNA, P. – BACH, A. – JUÁREZ, M. – DE LA FUENTE, M. A. 2008. Effect of a diet enriched in whole linseed and sunflower oil on goat milk fatty acid composition and conjugated linoleic acid isomer profile. J. Dairy Sci., vol. 91, 2008, p. 20-28. MEĽUCHOVÁ, B. – BLAŠKO, J. – KUBINEC, R. – GÓROVÁ, R. – DUBRAVSKÁ, J. – MARGETÍN, M. – SOJÁK, L. 2008. Seasonal variations in fatty acid composition of pasture forage plants and CLA content in ewe milk fat. Small Rumin. Res., vol. 78, 2008, p. 56-65. MEĽUCHOVÁ, B. – BLAŠKO, J. – KUBINEC, R. – GÓROVÁ, R. – MICHALEC, M. – VARGOVÁ, V. – KOVÁČIKOVÁ, Z. – MARGETÍN, M. – SOJÁK, L. 2009. Influence of floristic grazing cover on sheep milk quality. Acta fyt. et. zoot., vol. 12 (3), 2009, p. 57-64. NAGAO, K. – YANAGITA, T. 2005. Conjugated fatty acids in food and their health benefits. J. Biosci. Bioeng., vol. 100, 2005, p. 152-157. NUDDA, A. – MCGUIRE, M. A. – BATTACONE, G. – PULINA, G. 2005. Seasonal variation in conjugated linoleic acid and vaccenic acid in milk fat of sheep and its transfer to cheese and ricotta. J. Dairy Sci., vol. 88, 2005, p. 1311-1319. OSTROVSKÝ, I. – PAVLÍKOVÁ, E. – BLAŠKO, J. – GÓROVÁ, R. – KUBINEC, R. – MARGETÍN, M. – SOJÁK, L. 2009. Variation in fatty acid composition

Original paper of ewes‘ milk during continuous transition from dry winter to natural pasture diet. Int. Dairy J., vol. 19, 2009, p. 545-549. PETERSON, D. G. – KELSEY, J. A. – BAUMAN, D. E. 2002. Analysis of variation in cis-9, trans-11 conjugated linoleic acid (CLA) in milk fat of dairy cows. J. Dairy Sci., vol. 85, 2002, p. 2164-2172. SIGNORELLI, F. – CONTARINI, G. – ANNICCHIARICO, G. – NAPOLITANO, F. ORRÙ, L. – CATILLO, G. – HAENLEIN, G. F. W. – MOIOLI, B. – 2008. Breed differences in sheep milk fatty acid profiles: opportunities for sustainable use of animal genetic resources. Small Rumin. Res., vol. 78, 2008, p. 24-31. SOJÁK, L. – BLAŠKO, J. – KUBINEC, R. – GÓROVÁ, R. – ADDOVÁ, G. – OSTROVSKÝ, I. – MARGETÍN, M. 2012. Variation among individuals, breeds, parities and milk fatty acid profile and milk yield of ewes grazed on pasture. Small Rumin. Res. 2012, http://dx.doi.org/10.1016/j.smallrumres.2012.07.017, article in press. SOJÁK, L. – PAVLÍKOVÁ, E. – BLAŠKO, J. – MEĽUCHOVÁ, B. – GÓROVÁ, R. – KUBINEC, R. – EBRINGER, L. – MICHALEC, M. – MARGETÍN, M.2009. The quality of Slovak and Alpine milk products based on fatty health affecting compounds. Slovak J. Anim. Sci., vol. 42, 2009, p. 62-69. TSIPLAKOU, E. – KOMIAKIS, A. – ZERVAS, G. 2008. The interaction between breed and diet on CLA and fatty acid content of milk fat of four sheep breeds kept indoors or at grass. Small Rumin. Res., vol. 74, 2008, p. 179-187. TSIPLAKOU, E. – MOUNTZOURIS, K. C. – ZERVAS, G. 2006. The effect of breed, stage of lactation and parity on sheep milk fat CLA content under the same feeding practices. Livestock Sci., vol. 105, 2006, p. 162-167.

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Short communication

IMPACT OF THE PRESENCE OF MACROPHAGES IN RABBIT EJACULATES ON FEMALE FERTILITY

L. KUŽELOVÁ1, A. V. MAKAREVICH2 , P. CHRENEK1,2* Slovak University of Agriculture, Nitra, Slovak Republic Animal Production Research Centre Nitra, Slovak Republic

1 2

ABSTRACT The present study was designed to investigate the occurrence of macrophages in rabbit sperm and their impact on female fertility parameters in vivo. Activated macrophages, engaging in sperm phagocytosis (spermiophages), might represent a marker of innate immunosystem activation. Semen samples were collected from rabbit males using an artificial vagina, and spermiophages were identified with fluorescent dye Alexa Fluor 488-AcLDL (Acetylated Low Density Lipoprotein Molecular Probes, USA). For artificial insemination the sperm samples were divided into two groups basing on number of spermiophages: the group R1 - ejaculates with the occurrence of spermiophages less than 20 % of all cells and the group R2 - ejaculates with the occurrence of spermiophages at about 20 - 40 %. The increased occurrence of spermiophages, observed in the R2 group, was associated with decreased female fertility parameters (conceptional rate, liveborn pups). The differences in conceptional rates (73.08 vs. 68.18 %) as well as the differences in average number of liveborn pups per doe between groups of R1 and R2 does were not statistically significant. These preliminary results may indicate the negative impact of higher spermiophage presence in semen on female fertility. Key words: rabbit; spermatozoa; spermiophages; conceptional rate

INTRODUCTION Phagocytosis, the process by which cells engulf foreign particles, occurs in eukaryotes ranging from unicellular organisms, which use it for nutrition, to mammals, where it plays a key role in innate immunity (Gagnon et al., 2002). Leukocytes are present throughout the male reproductive tract, are found in most ejaculates, and are thought to play an important role in immune surveillance and phagocytic clearance of abnormal sperm (Gallegos-Avila et al., 2010). Polymorphonuclear (PMN) granulocytes account for 50 % to 60 % of all white blood cells (WBC) in semen, macrophages (MF) account for 20 % to 30 % and T-lymphocytes for 5 % (Smith et al. 1989; Wolff, 1995). Macrophages belong to the

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*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

mononuclear phagocyte system and they represent a mechanism to remove senescent, malformed or degenerative sperm. They appear to be involved in immunologic surveillance, immunoregulation, and tissue remodelling. Early morphological studies described the presence of macrophages in the interstitium and occasionally in the lamina propria of seminiferous tubules of human testis (Hermo et al., 1978; Pollanen et al., 1987; Frungieri et al., 2002). Phagocytosis of sperm by epithelial cells has been described previously in ductal tubules testis of various animal species (Dym, 1974; Hoffer and Hamilton, 1974; Hoffer et al., 1975; Holstein, 1978; Riva et al., 1981; Goyal, 1982). Spermiophagy by macrophages has been described in the male extragonadal ductal system (Holstein, 1978); in prostate and in boar

Received: October 25, 2012 Accepted: November 20, 2012

Slovak J. Anim. Sci., 45, 2012 (4): 144-147 ejaculate (Hrudka and Post, 1983). In cases of male infertility there has not been found a correlation with the percentage of abnormal spermatozoa in the semen or the motility of the spermatozoa and the number of spermiophage cells, in cases of necrozoospermia they were not dominant. Samples of semen voided after a prolonged period of abstinence did not show an increase in the spermiophage cells (Phadke, 1961). There is lack of information about occurrence of spermiophages in rabbit ejaculate and effect of spermiophages on female reproduction. The aim of the study was to identity spermiophages in rabbit ejaculates using specific staining and to evaluate the impact of spermiophagy on fertility of rabbit does.

MATERIAL AND METHODS Animals Sperm samples were obtained from sexually mature (4 – 5 months old) and clinically healthy rabbit males of M91 and P91 lines from the breeding farm of Animal Production Research Centre Nitra. The males were housed in individual cages, under a constant photoperiod of 14h of daylight. Temperature and humidity in the building were recorded continuously by means of a thermograph positioned at the same level as the cages (average relative humidity and temperature during the year were maintained at 60±5 % and 17±3°C). The rabbits were fed ad libitum with a commercial diet (KV; TEKRO Nitra, s.r.o.) and water was provided ad libitum with nipple drinkers. Analysis of spermiophages The ejaculates of sperm were washed in a saline solution. Fetal calf serum (FCS) was added and the samples were centrifuged at 1000 rpm for 3 min to separate it from the rest of seminal fluid. Pellets were resuspended in 2 µg.ml-1 of Alexa-AcLDL in a saline solution, added with FCS and incubated in incubator for 2 – 4 hours. The samples were subsequently centrifuged at 1000 rpm for 3 min and resuspended in a cold saline solution. The suspension was afterwards placed onto a microscope slide, mixed with an equal volume of Vectashield antifade medium (Vector Laboratories, Burlingame, CA) containing DAPI fluorochrome. The drop was covered with a coverslip. Samples were evaluated under a Leica fluorescent microscope (Leica Microsystem, Germany). If the LDL (Low-Density Lipoprotein Complexes) has been acetylated, the LDL complex no longer binds to the LDL receptor, but rather is taken up by spermiophages (macrophages) that possess “scavenger” receptors specific for modified LDL. The superior fluorescence output by Alexa Fluor AcLDL provides easier identification

Short communication of spermiophages and endothelial cells in mixed cell population. Insemination Sexually mature and clinically healthy rabbits does (n=48) that were included in the rearing programme were used for artificial insemination. Spermatozoa were diluted in a commercial diluent (MiniTüb, Tiefenbach, Germany) up to the concentration minimum of 14x106/ml and used for the insemination of hormonally stimulated rabbit females. Females of rabbits were inseminated with fresh doses of filtered heterospermic semen (0.5ml I.D. per female), divided into two groups R1 (n=2) and R2 (n=2). PMSG at 25 I.U. (Sergon, Bioveta, Czech Republic) was administrated to each doe 48 hours before artificial insemination (A.I.). Immediately following AI, synthetic GnRH (2.5 μg; Supergestran, FerringPharmaceuticals, Czech Republic) was intramuscularly injected into each doe. The ratio of kindled does to the number of inseminated does (kindling rate) and also the average number of liveborn kits per 1 inseminated doe were recorded. Conceptional rate was determined basing on pregnancy diagnostics on 17th day following insemination. Obtained data were statistically processed by χ2 –test using Excel software.

RESULTS AND DISCUSSION According to spermiophage occurrence in sperm ejaculates the samples were divided into two groups: the ejaculates with the occurrence of spermiophages (SP) less than 20 % of all cells - the R1 group, and the ejaculates with the occurrence of spermiophages in the range of 20-40 % - the R2 group (Fig. 1). Results of insemination trials showed that higher occurrence of spermiophages led to decrease in female fertility parameters: conceptional rate and liveborn pup rate compared to the group with less count of spermiophages (R1). However, the differences in conceptional rates and in average number of liveborn kits per doe between R1 and R2 groups were not statistically significant. Values of fertility parameters within the groups R1 and R2 are shown in Table 1. Tomlinson et al. (1992) suggested the possibility that macrophages may play a positive role in the control of semen quality. Specifically, the authors postulated that phagocytes might shape the quality of the human ejaculate by phagocytosing morphologically abnormal spermatozoa. Tomlinson et al. identified three types of seminal phagocytic cells: small PMN leukocytes, monocytes of similar size, and much larger macrophages capable of engulfing multiple sperm heads. These authors suggested

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Slovak J. Anim. Sci., 45, 2012 (4): 144-147

Fig. 1: a) Sperm population stained with nuclear fluorochrome DAPI b) Colony of macrophages specifically stained with Alexa-AcLDL fluorescent dye

Table 1: Impact of spermiophage occurrence in male ejaculates on female fertility in vivo Rabbit No. No. Conceptional Kindling males inseminated fertilized rate % rate % females females

Liveborn pups (n)/ average per doe (%)

Stillborn pups (n)/ average per doe (%)

Group R1 (n=2)

Group

R2 (n=2)

73.08

69.23

(182)/9.58

(9)/0.47

68.18

(105)/7.00

(4)/0.27

that the sperm morphology is directly correlated with the size of the seminal leukocyte population (GallegosAvila et al., 2010). Chrenek et al. (2010) evaluated sperm viability of rabbit males using fluorescent analysis (SYBR14/PI and annexin V/DAPI tests). The present results show that female fertility parameters were decreased in group with higher occurrence of spermiophages, but these differences were not statistically significant. Once seminal leukocyte concentration rises above a threshold of 1x106â &#x201E;ml (leucocytospermia), they have significant potential to damage sperm and cause infertility (Plante et al., 1994; Wolff, 1995; Ochsendorf, 1999; Sharma et al., 2001; Henkel et al., 2003). The presence of spermiophages was associated with lower total sperm count and sperm concentration, lower forward motility and high percentage of disrupted sperm compared to

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ejaculates without spermiophages (Pelliccione et al., 2008). These authors showed that spermiophages engulfing sperm are frequently observed in ejaculates from non-leukocytospermic men complaining for couple infertility.

CONCLUSION Our data suggest that spermiophages might have a negative biological impact on female fertility regardless to their count in semen. Since these are preliminary and not statistically significant results, therefore further experiments are required in order to prove the impact of spermiophages on doeâ&#x20AC;&#x2122;s reproductive traits.

Slovak J. Anim. Sci., 45, 2012 (4): 144-147 ACKNOWLEDGMENT This work was supported from the grants of Slovak Research and Development Agency: APVV0566-011.

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List of reviewers – Volume 45 Editorial board of the Slovak Journal of Animal Science thanks all reviewers of manuscripts for their cooperation.

R. Omelka M. Oravcová V. Parkányi J. Pivko J. Rafay Ľ. Rajčáková N. Sasáková A. Sirotkin L. Soják F. Strejček P. Suchý M. Šimko V. Tančin J. Tomka J. Trandžík M. Uhrinčať J. Vašíček K. Vašíčková M. Vršková

D. Apolen H. Arpášová V. Brestenský M. Čanigová Z. Čerešňáková J. Čuboň E. Hanusová L. Hetényi E. Horniaková P. Chrenek P. Juhás R. Jurčík K. Kirchnerová J. Kopernický J. Kottferová E. Kubovičová A.V. Makarevič M. Margetín M. Milerski L. Molnár

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Slovak J.Anim.Sci., 45, 2012 (4) Contents

Original papers KADLEČÍK, O. – PAVLÍK I.: Genealogical analysis in small populations: the case of four Slovak beef cattle breeds

111

ŠŤASTNÁ, D. – ŠŤASTNÝ, P.�: Efficiency of treatment of follicular cysts in cows

118

KULÍKOVÁ, B. – SLAMEČKA, J. Jr. – MAKAREVICH, a. v. – CHRENEK, P.: Effect of in vitro culture conditions on the quality of rabbit embryos

123

PAYA, h. – TAGHIZADEH, a. – LASHKARI, s. – SHIRMOHAMMADI, s.: Evaluation of rumen fermentation kinetics of some by-products using in situ and in vitro gas production technique

127

SOJÁK, l. – KUBINEC, r. – BLAŠKO, j. – ČABALA, r. – LORENZ, w. – APOLEN, d. – MARGETIN, m.: Quality improvement of Slovak ewe milk based on the content of health affecting fatty acid compounds

134

Short communication KUŽELOVÁ, L. – MAKAREVICH, A. V. – CHRENEK, P.: Impact of the presence of macrophages in rabbit ejaculates on female fertility�

ISSN 1337-9984 (Print) ISSN 1338-0095 (Online)

144