Reproductive problems and their association with chromosomal anomalies in Dairy cattle and buffaloes

Introduction

Reproduction is a complex physiological process, regardless the species, breed or sex of the individual. Reproduction in all the species and breeds of animals is affected by genetics or environment as such and can lead to interruption in reproductive processes at any stage in gametogenesis, during transportation of the gametes to the site of fertilization or during the fetal, perinatal or neonatal growth phases. Animal geneticists and veterinarians have advanced in animal cytogenetic studies to predict the potential role of genes in reproductive defects in domestic animals. This development stems from advances and recognition in human genetics over the last century, highlighting that many reproductive problems may have a genetic basis. Hereditary factors play an important role in infertility or sub-fertility caused in animals. It has been well-recognized that numerical and structural chromosomal aberrations can lead to reproductive disturbances not only in mammals but also in higher plants (Ford, 1982) [1] . An area where the potential of veterinary cytogenetics can be fully exploited is reproduction since an inherited aberration that affects reproductive efficiency can hinder the success achieved over the years in animal breeding. Reproductive disorders occur frequently in lactating dairy cows and can dramatically affect reproductive efficiency in a dairy herd. Some of the most common disorders include ovarian cysts, freemartins, early embryonic loss and retained placenta. These are diverse disorders that result in impaired reproductive function Fricke et al (2007) [2] . The role of chromosomal abnormality as a cause of impaired reproductive function is crucial and is often associated with infertility in the carrier, early mortality of embryos and newborns, underdevelopment and degeneration of reproductive organs, poor semen quality, and decreased weight gain in young, as well as functional and, more rarely, phenotypic disturbances Ioana (2007) [3] . Dairy heifers rearing and management requires substantial financial investment. However, many times heifers turn out infertile or sub-fertile, which lead to hindrance to profit in cattle and buffalo oriented dairy industry. Possible losses due to reproductive problems are in terms of cost of rearing heifer till puberty, their lifetime production and future output from calves. The productivity of cattle largely depends on their reproductive performance. For this reason, keeping in mind that it is necessary to keep breeding animals under cytogenetic control, particularly in cattle and buffalo population, the study was designed and incidence of reproductive problems in five years on an organized farm was calculated and its association with chromosomal anomalies was drawn.

Material and Methods

A. Study of Incidence of reproductive problems

All the experimental procedures involving animals were approved by the Institutional Animal Ethics Committee (IAEC), National Dairy Research Institute, Karnal, India.

Incidence of reproductive problems in Sahiwal (SW) and Karan Fries

A. Criteria for selection

Animals calved in a particular year were studied for incidence of reproductive problems in the same year. Animal record was searched and information gathered from the date of calving. The incidence of post-partum anoestrus in cows which failed to return to oestrus more than 50 days postpartum in cattle and 90 days in buffalo was recorded. Incidence of ovarian cyst either follicular or luteal cyst were recorded as reported in the daily AI register and year wise auction list of animals at Livestock Research Station (LRS) in cows. Incidence of delayed puberty was recorded in heifers that did not exhibit oestrus behaviour after 30 months in cattle and 35 months in buffaloes. Incidence recording for repeat breeder syndrome was based on the number of services after calving. Those cows and heifers failed to conceive after 4 AI were recorded as repeat breeders in the herd. In order to compare the problems a control group was selected by different criteria. It comprised of each breed with normal calving in the same year, without the history of anoestrus, abortion and irregular oestrus cycle. Control females for postpartum anoestrus were contemporaries that conceived in 50 and 90 days of the postpartum period in cattle and buffalo, respectively. In case of repeat breeder incidence study, control animals were contemporaries who conceived in less ≤4 AI. A control group for delayed puberty in heifers were animals that attend puberty before 30 months in the case of cattle and before 35 days in case of buffaloes

B. Cytogenetic studies

The animals for the proposed investigation were selected as abnormal (experimental) and normal (control) groups belonging to two species of dairy animals, viz., cattle and buffaloes. In total about 50 problematic animals were identified from each breed of cattle and buffalo i.e. Sahiwal (SW), Karan Fries (KF) and Murrah (MU), respectively. Similarly from each breed of cattle and buffalo normal (control) animals (10 per breed) were chosen at random from the existing herd. The criterion for selection was the females with a record of three successive normal calving and completion of lactations each of 305 days or more. Thus, a total of 180 animals were investigated cytogenetically for different numerical and chromosomal anomalies. In general, well-spread 50 Giemsa-stained metaphase plates were screened for each animal; however, in the case of some anomaly, 100 or more metaphases were examined. Slides were further subjected to C-and R-Banding methods for the identification of abnormal chromosomes.

Chromosome preparations:Blood sample of about 10 ml under strict aseptic care was collected in vacutainer tubes. Blood was brought to the laboratory and was stored in the refrigerator at 2-8 °C till used (1-3 days). Whole blood was used for setting up cultures according to the method described Yadav and Balakrishnan (1985) [4] . An aliquot of 0.5 ml blood was added to each culture glass bottle of 30 ml capacity containing 6 ml medium consisting of RPMI 1640, bovine foetal calf serum (10%), phytohaemaglutinin (10µg/ml) and penicillin streptomycin (20 IU/ml). The cultures were incubated for 96 hours at 37.5±0.5 °C . Seven hours before harvesting of cultured cells, Bromo-deoxy-uridine (BrdU, 20 mg/ml) was added to each culture bottle. Subsequently, 45 minutes before harvesting of cultured lymphocytes drops (10 µg/ml) of colchicine solution was added to each culture bottle. Cultures were harvested with the routine method of hypotonic solution treatment and fixation with methanol: glacial acetic acid (3:1). Chromosome preparations were made on glass slides using the air-dried method. Slides were stained with Giemsa (2% at pH 6.8) dye for conventional chromosome examination. Evaluation of a total of 50 metaphase spreads in each animal was done. To identify the anomaly slides were subjected to obtain R-banding with RB-FPG technique Mukherjee and Yadav (2004) [5] and C-banding with BSG method Sumner (1972) [6] . Details of band patterns of autosomes and X chromosome were compared and matched with standard karyotype to identify chromosome involved in mosaicism.

C. Statistical analysis

Statistical analysis using SAS 9.2 software was done for analysis of data obtained through reproduction records. Frequency of gaps, chromatid break, chromosomal break and X chromosome fragility was identified in 50 metaphase plates per animal in 50 experimental and 10 control animals, of the SW and KF cattle breed and MU buffalo breed. Among 2500 metaphase plates examined in 50 animals of each breed overall frequency of chromatid and chromosome break was determined. Per cent distribution of fragile sites in SW, HF and MU breed abnormal and normal groups was calculated. Mean values and standard deviations of chromosome abnormalities in cells of abnormal and control groups of each breed were estimated and the F test between abnormal and control was applied to determine significance using F values of the observed difference in the mean rate of X chromosome fragility, chromatid break/cell, chromosome break/cell and gap/cell within abnormal and control group comparison in each breed. Animal # Mu 6087, 3.5 years old heifer when screened cytogenetically with no observed oestrus behaviour showed XO condition with presence of 10 pairs of submetacentric autosomes, 38 acrocentric autosomes and one large acrocentric X chromosome in all the metaphase plates examined as depicted in figure 8. The heterochromatin specific C-banding technique used to identify autosomes and sex chromosomes showed one X chromosomes with prominent typical triangular centromeric band along with a thin intercalary band specific to buffalo (Figure 9) confirming the XO condition in buffalo heifer.

Results

In the control group of animals no numerical anomaly was observed. Karyotype prepared for normal animals consisted of 29 pairs of acrocentric autosomes and one pair of submetacentric sex chromosomes in cattle. In buffalo karyotype comprised of pairs [1][2][3][4][5] of submetacentric autosomes, rest 20 pairs were acrocentric with sex chromosomes being longest one among them. Animal calved a male calf on 26.11.2013, however, it was disposed young and was not available for karyotyping. Cytogenetic screening of giemsa stained 100 metaphase plates was done and karyotypes were prepared which confirmed presence of translocation in 100% plates. The heterochromatin specific CBG banding technique used to identify autosomes and sex chromosomes showed two submetacentric chromosomes of equal length in which centromeric band was absent. This observation debarred the possibility of occurrence of balanced reciprocal X-autosomal translocations. Further, R banding was done and chromosomes were matched and arranged according to standard cattle karyotype (Fig. 101112).

B.2 Structural chromosome anomalies in SW and KF cattle breeds and MU buffalo breed

Discussion

The incidence of reproductive problems in cows and heifers of each breed cattle and buffalo were recorded for five years. SW cows, showed the highest incidence of anoestrus (19.6%), followed by repeat breeding (12.6%) and abortions (8.40%). KF breed showed a trend as the highest incidence of anoestrus (21.12%), followed by abortions (11.75%) and repeat breeding (11.61%). MU buffalo cows reported highest incidence of anoestrus (31.46%), followed by repeat breeding. Sarder et al (2010) [7] reported 20.4% incidence of anestrus in cattle followed by repeat breeding (12.8%); Serur et al (1982) [8] reported the higher prevalence of anestrus (49.9%) followed by repeat breeding (15.1%). However, the highest incidence of repeat breeding was shown in several studies. Khair et al (2013) [9] in cattle showed highest cumulative incidence of repeat breeding and lowest incidence of abortions. Rabbani et al.

(2010) [10] reported 15.69% repeat breeding followed by 9.74 % anoestrus and 5.99% abortion prevalence in buffaloes. El-Bayomi et al. (2011) [11] showed that the percent of total numerical aberrations for repeat breeder group was 19.95%, for vaginal prolapse group was 13.6% and for cows suffering from anoestrum was recorded as 21%. These results were significantly higher than that observed in the control group.

Sex chromosome mosaicism such as 60, XY / 61, XYY in cattle was reported by Krumrych et al. (2002) [12] , 59, XO/60, XX/61, XXX mixoploid in Pinzgauer x Angus heifer and 59, XO/ 60, XX/ 61, XO (with iso-X) mixoploid in Charolais was reported by Swartz and Vogt (1983) [13] . Another animal MU 6087 had 49, XO condition known as Turner's syndrome. Yadav and Balakrishnan (1982) [14] reported aneuploidies such as X chromosome trisomy in a ten year old buffalo and X chromosome monosomy (Yadav et al., 1990; Prakash et al., 1992; Iannuzzi et al., 2000) [15,16,17] have been reported in farm animals.

Structural chromosome anomalies in SW and KF cattle breeds and MU buffalo breed

Structural anomalies with an increased percent were reported. El-Bayomi et al. (2011) [11] showed that the percent of total structural aberrations for repeat breeder group were 62% and vaginal prolapse it was 40% which were higher in comparison to control group as shown in present study.

Fragile sites are specific chromosome regions, which appear as nonstaining gaps or breaks in one or both chromatids, not in all metaphase spreads. The chromosome fragility on the autosomes and X chromosome (BTAX) has been associated with reproductive abnormalities, bald calf syndrome, and dwarfism (Lambi and Postiglioni, 1996) [18] . The present study identified the higher frequency of gaps and breaks in the abnormal group (consisting of anoestrus, repeat breeding, and abortion cases) compared to control animals (Table 7 and8). Farag et al. (1999) [19] studied chromosomal fragility in different groups of reduced fertility, such as anoestrus and repeat breeding, other groups comprising uterine prolapse, heterosexual and isosexual twins which showed that the structural and numerical aberrations of the chromosomes were more frequent in abnormal females of cattle and buffaloes than the control groups. In cattle, the females with cystic ovary had the highest percentages of the chromosomal gaps and deletions (in autosomes and sex chromosomes), whereas the repeat breeder cases had the highest rates of chromatid breaks and centric fusions (in autosomal chromosomes). In buffaloes, the repeat breeder females had the highest proportion of the chromosomal gaps (especially in autosomal chromosomes). The F values for X chromosome fragility, chromatid break, chromosome breaks and gaps were statistically significant (P<0.001) between control and abnormal group as shown in table 12, 13 and 14. These findings are in accordance with El Bayomi et al. (2011) [11] who reported percent of total structural aberrations were predominant and reached 62%, these results were significantly higher than observed for control group of repeat breeding animals. Saleh (2008) [20] reported a highly significant percent of gaps, breaks and fragments for aonestrum group. Farag et al. (1999) [19] showed that the structural and numerical aberrations of the chromosomes were more frequent in abnormal females of cattle and buffaloes than the control groups. They reported that in cattle, the females with cystic ovary(ies) had the highest percentages of the chromosomal gaps and deletions (in autosomes and sex chromosomes), whereas the repeat breeder cases had the highest rates of chromatid breaks and centric fusions (in autosomal chromosomes). In buffaloes, the repeat breeder females had the highest proportion of the chromosomal gaps (especially in autosomes). Maity et al.

(1996) [21] recorded a highly significant percent for breaks 15% in cows suffering from repeat breeding and explained that gaps and breaks either in chromatid or chromosomes play an important role in fertility problems. The reproductively abnormal animals with higher percentage of chromosomal anomalies may be due to losses of part of chromosomes carrying genes responsible for reproduction, increasing their frequencies accompanied mostly by losses of genes responsible for fertility performance. Hassanane et al. (1995) [22] reported that chromatid gaps and breaks are related to anoestrum with reduced fertility in animals. De Hondt et al. (1988) [23] and El Nahass et al. (1974) [24] found high percentages of cells with gaps or secondary constriction in infertile cattle. [25] reported case of X autosome translocation i.e., reciprocal translocation to be completely sterile while almost uninterrupted fertility was noted, initially, in an X-autosome translocation carrier cow identified in the United States (Eldridge, 1985) [26] . Basrur et al. (2001b) [27] reported X-AT carriers resulted in high rates of return to oestrus between days 28 and 60, abortions between days 121 and 235 after insemination; similar findings were witnessed in the present case. SW 1985 was a repeat breeder with return to oestrus in 30-45 days after insemination, which indicated resorbption of postfertilization zygotes or early embryos (Gustavsson, 1980; Popescu, 1990) [28,29] . The problematic female in the present study was morphologically and gynaecologically normal as revealed on per rectum examination, similar findings were reported by Di meo et al. (2011) [30] in a five year old buffalo having centric fusion translocation. The female carrier had normal body conformation and internal sex adducts.

Conclusion

The present study in reproductively problematic animals showed a wide range of anomalies including numerical and structural ones. These were identified only after recording problematic cases with prolonged history of reproductive problems such as anoestrus, repeat breeding, abortions and irregular cycles. It has been found that animals were treated many times by veterinarians, even then the occurrence remained the same, which emphasizes that the over expenses in terms of veterinary aid, management can be avoided by cytogenetic screening at an early age.

Acknowledgement

Acknowledgements 1. Author is thankful to NDRI for all the necessary funding. 2. Ford, C. E., Structural rearrangements and infertility in mammals. In Proceeding of the Fifth European Colloquium of Cytogenetics in Domestic Animals, Milano-Gargnano, Italy, 7-11 June1982, pp. 17-41.

References

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28. Di Meo, G. P., Perucatti, A., Genualdo, V., Iannuzzi, A., Sarubbi, F., Caputi-Jambrenghi, A, Incarnato, D, Peretti, V, Vonghia, G and Iannuzzi, L., A rare case of centric fission and fusion in a river 32. buffalo (Bubalus bubalis=50) cow with reduced fertility. Cytogenet. Genome Res., 2011, 132 [1-2] , 26-30
29. Ford CE. Structural rearrangements and infertility in mammals. In Proceeding of the Fifth European Colloquium of Cytogenetics in Domestic Animals, Milano-Gargnano, Italy, 7-11 June1982. 1982:17-41.
30. Fricke MP, Revay T, Saver DR. Managing Reproductive Disorders in Dairy Cows. Cytogenet Genome Res. 2007;116:80-84.
31. Ioana-Nicolae. The role of cytogenetics in the genetic improvement of cattle. In 3rd Joint Meeting of the Network of Universities and Research Institutions of Animal Science of the South Eastern European Countries, Thessaloniki. 2007.
32. Yadav BR, Balakrishnan CR. Parallelism of XX/XY Chimaerism in cattle and buffalo multiple birth. Indian J of Dairy Sci. 1985;38:62-65.
33. Mukherjee S, Yadav BR. Differential staining (RB-FPG) patterns of sex chromosomes of some breeds of zebu and crossbred cattle. Indian J Dairy Sci. 2004;57:412-415.
34. Sumner AT. A simple technique for demonstrating centromeric heterochromatin. Exptl Cell Res. 1972;75:304306.
35. Sarder MJU, Moni MIZ, Aktar S. Prevalence of reproductive disorders of cross breed cows in the Rajshahi district of Bangladesh. SAARC J Agri. 2010;8:65-75.
36. Serur BH, Farrag AA, Gomma A. Incidence of certain infertility problems among cows and buffaloes in upper, Egypt. Assiut Vet Med J. 1982;10:209-214.
37. Khair A, Alam MM, Rahman AK, Islam MA, Azim MT, Chowdhury EH. Incidence of reproductive and production diseases of cross-bred dairy Cattle in Bangladesh. Bangl J Vet Med. 2013;11(1):31-36.
38. Rabbani RA, Ahmad I, Lodhi LA, Ahmad N, Muhammad G. Prevalence of various reproductive disorders and economic losses caused by genital prolapse in buffaloes. Pakistan Vet J. 2010;30(1):44-48.
39. El-Bayomi KM, El-Araby IE, Zaglool AW. Cytogenetic analysis related to some infertility problems in cattle. Glob Veterinaria. 2011;7(4):323-329.
40. Krumrych W, Jędrzej M, Jaśkowski MG, Janusz Z. First case of 61,xyy-trisomy in bull in Poland -initial observations. Bull Vet Inst Pulawy. 2002;46:307-315.
41. Swartz HA, Vogt DW. Chromosome abnormalities as a cause of reproductive inefficiency in heifers. J Hered. 1983;74:320-324.
42. Yadav BR, Balakrishnan CR. Trisomy of X-chromosome in Murrah buffalo. Vet Rec. 1982;111:184-185.
43. Yadav BR, Kumar P, Tomer OS, Kumar S, Balain DS. Monosomy X and gonadal dysgenesis in a buffalo heifer (Bubalus bubalis). Theriogenol. 1990;34:99-105.
44. Prakash B, Balain DS, Lathwal SS. A 49,X0 sterile Murrah buffalo (Bubalus bubalis). Vet. Rec. 1992;130:559-560.
45. Iannuzzi L, Di Meo GP, Perucatti A, Incarnato D, Schibler L, Cribiu EP. Comparative FISH mapping of bovid X chromosomes reveals homologies and divergences between the subfamilies Bovinae and Caprinae. Cytogenet. Cell Genet. 2000;89:171-176.
46. Lambi S, Postiglioni A. Frequencies and cytomorphological manifestation of sexual X-chromosome fragility (FRAXq 3.1) in Holstein-Friesian. Arch. Zootec. 1996;45:203-208.
47. Farag LM, Abd EI-Gawad EMM, EI-Naliass E, Hassan AGM, Abd Allah SM. Chromosome anomalies in cattle and buffaloes in relation to reduced fertility and uterine prolapse with reference to early detection of chromosome anomalies in calves. J. Egypt, Ger. Soc. Zool. Hist. and Histochem. 1999:219-232.
48. Saleh AA. Cytogenetic studies related to some reproductive disorders in cattle. Master thesis, Fac. Vet. Fac. of Vet. Medicine. Zagazig Univ. 2008.
49. Maity BA, Sahoo K, Ghaudhuri G. Cytogenetic studies on the Taurusindicus crossbred cows of poorer breeding status. Ind J Anim. 1996;12.
50. Hassanane MS, Mahrous KF, Abdoon ASS, Gadallah F. A case report: Mosaic diploid/ tetraploid in a pregnant cow infected with lumpy skin disease. Vet. Med. J. Giza. 1995;43:137-140.
51. De Hondt HA, El-Nahass E, Fathy K. Cytogenetic studies on Egyptian native cattle and some of its crossbreds. J. Anim. Bred. Genet. 1988;105:400.
52. El-Nahass E, Syrjalla A, Michelmann HW, Paufler S. Mosaik einer Xchromosome aberration als wahrscheinliche Ursache der Sterilitat bei einem Rind. Dtsch Tie-roerztl Wochenschr. 1974;81:397-398.
53. Gustavsson I, Fraccaro M, Tiepolo L, Lindsten J. Presumptive X-autosome translocation in a cow: preferential inactivation of the normal X chromosome. Nature. 1968;218:183-184.
54. Eldridge FE. Cytogenetics of livestock. Westport, Connecticut: AVI Publishing Company, Inc.; 1985:79-85 & 115-163.
55. Basrur PK, Reyes ER, Farazmand A, King WA, Popescu PC. X autosome translocation and low fertility in a family of crossbred cattle. Anim. Reprod. Sci. 2001;67(1-2):1-16.
56. Gustavsson I. Chromosomal aberrations and their influence on the reproductive performance of domestic animals-a review. Heredites. 1980;68:331-332.
57. Popescu CP. Chromosomes of the cow and bull. In: McFeely RA, ed. Domestic Animal Cytogenetics. San Diego: Academic Press; 1990:41-66.
58. Di Meo GP, Perucatti A, Genualdo V, Iannuzzi A, Sarubbi F, Caputi-Jambrenghi A, et al. A rare case of centric fission and fusion in a river buffalo (Bubalus bubalis=50) cow with reduced fertility. Cytogenet. Genome Res. 2011;132(12):26-30.