Short communication: Validation of in vitro fertility genes in a Holstein bull population.

Previously, we constructed an in vitro fertilization system for the identification of genes affecting fertility traits in dairy cattle. The efficiency of this system has been demonstrated by the identification of several genes affecting fertilization rate and early embryonic survival. However, to employ these genetic markers in marker- and gene-assisted selection programs, there is a need to validate in vitro results in phenotypic data sets collected in vivo. Thus, the objective of this study was to validate, in a population of Holstein bulls, the fertility trait genes we previously identified in an in vitro system. Estimated relative conception rate (ERCR) data from 222 Holstein bulls were obtained from 5 different artificial insemination companies in the United States. Bulls were genotyped for the genes FGF2, POU1F1, PRL, PRLR, GH, GHR, STAT5A, OPN, and UTMP, and the data were analyzed for association with ERCR using a mixed effects sire model. A stepwise model selection procedure revealed evidence of association with ERCR for FGF2 and STAT5A polymorphisms. The in vivo validation suggests that these genes can be used in gene-assisted selection programs for reproductive performance in dairy cattle. The genotypes found to be associated with low bull fertility in this study have been reported to be associated with high milk composition in previous studies. These findings provide molecular evidence for the antagonistic relationship between milk production and fertility observed for many years in different breeds of dairy cattle.

Analysis of segregation distortion and association of the bovine FGF2 with fertilization rate and early embryonic survival.

Fibroblast growth factor 2 (FGF2) plays an important role in fertility and early embryo development. The objectives of this study were to test the association of FGF2 polymorphisms with fertilization success in cattle using an in vitro fertilization experimental system and to investigate the mechanisms leading to the presence of rare alleles of FGF2 in the Holstein population. A total of 7502 fertilizations were performed and a total of 5049 embryos were produced to collect fertilization and embryo survival records. A total of 444 ovaries, from which oocytes were aspirated and fertilized, were genotyped for two single nucleotide polymorphisms (SNPs) previously identified in FGF2 (g.23G>T and g.11646A>G). Frequency of the TT genotype of the g.23G>T SNP was low in the ovary population (5.4%) and in a different Holstein cattle population (6.6%) examined in this study. Single SNP analysis showed that both SNPs were associated with early embryonic survival rate. Two-way interaction analysis revealed significant association of epistatic interaction between the SNPs with fertilization rate. To test whether or not low frequency of allele T for the g.23G>T SNP in the population is a result of a fertilization failure of T oocytes, semen from six GG bulls was used to fertilize a total of 458 oocytes obtained from 19 GT ovaries. A significant segregation distortion was observed for 169 embryos genotyped for the g.23G>T SNP. We conclude that oocytes carrying the T allele show a reduced fertilization rate and that segregation distortion leads to rarity of the TT genotype in the population.

Effects of signal transducer and activator of transcription (STAT) genes STAT1 and STAT3 genotypic combinations on fertilization and embryonic survival rates in Holstein cattle.

Infertility is a major cause of dairy cow culling and economic loss. Signal transducer and activator of transcription (STAT) proteins are transcription factors that play an important role in fertility and early embryonic development, among many other functions. Previous studies have reported the association of several genes from the JAK/STAT signaling pathway with fertility traits in cattle. The STAT1 and STAT3 genes are members of this pathway and are known to interact with each other by forming a heterodimer complex that enters the nucleus and controls expression of specific genes. Thus, the objective of this study was to investigate the effects of the interactions between polymorphisms in these genes on fertilization and early embryonic survival rates using an in vitro fertilization system. A total of 7,519 oocytes, collected from 445 ovaries, were exposed to sperm and a total of 5,075 embryos were produced. Fertilization rate was calculated as the number of cleaved embryos at 48 h post-fertilization out of the total number of oocytes exposed to sperm. Early embryonic survival rate of embryos was calculated as the number of blastocysts on d 7 of development out of the total number of embryos cultured. Effects of ovary genotypes on fertilization and early embryonic survival rates were evaluated. Single-SNP analysis revealed a statistically significant association between SNP25402 in STAT3 and fertilization rate. Oocytes produced from ovaries with AA genotype showed a 0.701 fertilization rate versus 0.666 and 0.663 for oocytes produced from AC and CC ovaries, respectively. The interaction between STAT3 SNP (SNP19069/SNP25402) was highly significant for survival rate but not for fertilization rate. Also, the interaction between STAT1 SNP and SNP19069 was highly significant for survival rate. Genotype combinations found to promote fertilization and embryonic survival could be incorporated into breeding programs aimed at improving fertility performance in dairy cattle.

Single gene and gene interaction effects on fertilization and embryonic survival rates in cattle.

Decrease in fertility and conception rates is a major cause of economic loss and cow culling in dairy herds. Conception rate is the product of fertilization rate and embryonic survival rate. Identification of genetic factors that cause the death of embryos is the first step in eliminating this problem from the population and thereby increasing reproductive efficiency. A candidate pathway approach was used to identify candidate genes affecting fertilization and embryo survival rates using an in vitro fertilization experimental system. A total of 7,413 in vitro fertilizations were performed using oocytes from 504 ovaries and semen samples from 10 different bulls. Fertilization rate was calculated as the number of cleaved embryos 48 h postfertilization out of the total number of oocytes exposed to sperm. Survival rate of embryos was calculated as the number of blastocysts on d 7 of development out of the number of total embryos cultured. All ovaries were genotyped for 8 genes in the POU1F1 signaling pathway. Single-gene analysis revealed significant associations of GHR, PRLR, STAT5A, and UTMP with survival rate and of POU1F1, GHR, STAT5A, and OPN with fertilization rate. To further characterize the contribution of the entire integrated POU1F1 pathway to fertilization and early embryonic survival, a model selection procedure was applied. Comparisons among the different models showed that interactions between adjacent genes in the pathway revealed a significant contribution to the variation in fertility traits compared with other models that analyzed only bull information or only genes without interactions. Moreover, some genes that were not significant in the single-gene analysis showed significant effects in the interaction analysis. Thus, we propose that single genes as well as an entire pathway can be used in selection programs to improve reproduction performance in dairy cattle.

Mutations in the STAT5A gene are associated with embryonic survival and milk composition in cattle.

The objective of this study was to investigate the association of the signal transducer and activator of transcription 5A (STAT5A) gene with fertilization rate, embryonic survival, and milk production and composition in cattle. The STAT proteins are transcription factors that are specifically activated to regulate gene transcription when cells encounter cytokines and growth factors. The STAT5A gene is a member of the interferon-tau (IFN-tau) and placental lactogen (PL) signaling pathway, which is involved in both milk production and initiation of pregnancy. Using the DNA-pooling sequencing approach, a total of 12 single nucleotide polymorphisms (SNP) were identified, 1 exonic and 11 intronic. For the study of association of these SNP with embryonic survival, 1,551 embryos were produced in vitro from 160 cows and 3 sires. Significant associations with embryonic survival were found for 7, 5, and 2 SNP for embryos produced from sires 1, 2, and 3 respectively. The association of fertilization rate with STAT5A polymorphisms was evaluated in more than 2,300 oocytes. Significant associations were found for 6, 2, and 2 SNP for sires 1, 2, and 3 respectively. For sire 1, 5 SNP showed significant associations with both embryonic survival and fertilization rate compared with 1 SNP for sires 2 and 3. To determine if embryonic losses had occurred before the blastocyst stage, 145 of the surviving embryos were harvested at d 7 of development and genotyped for the single exonic SNP12195. A significant segregation distortion was observed between oocytes produced from 2 sires carrying the same genotype. Thus, it is most likely that STAT5A is associated with 2 mechanisms of embryo death. One is a prefertilization mechanism involving sperm factors that cause low fertilization rate. The second is a postfertilization mechanism that causes incompatibility between the male pronucleus and the oocyte, which in turn leads to death of the embryo before the blastocyst stage. Association testing of SNP12195 (exon 8) and SNP14217 (intron 9) with milk composition revealed that allele G of SNP12195 was associated with a decrease in both protein and fat percentages. However, SNP14217 in intron 9 showed no significant association with milk production or health traits. The G allele of SNP12195 was also associated with low embryonic survival, making this SNP an attractive candidate for progeny testing programs in dairy cattle.

Pattern of expression of the uterine milk protein gene and its association with productive life in dairy cattle.

The uterine milk proteins (UTMP) are the major proteins secreted by the endometrium, primarily under the control of progesterone. Specific functions of UTMP are poorly understood, but may include protease inhibition, nutrition of the conceptus, growth control, and suppression of the maternal immune system. The uterine milk protein gene (UTMP) was chosen for this study because of its possible roles in health traits and results of previous studies on the association of the UTMP region with milk production and productive life in dairy cattle. Expression of UTMP was examined in 198 bovine tissues obtained from 10 fetuses and 17 cows. Transcripts of UTMP were found in all cotyledon tissues examined and to a lesser extent in ovary, pituitary, and spleen tissues obtained from fetuses. The UTMP gene was predominantly expressed in endometrium (17/17), ovary (15/16), and caruncle (12/12) tissues obtained from cows. The predominant expression of UTMP in reproductive tissues is consistent with an important role of this gene in reproductive success. To investigate the association between UTMP and production traits in cattle, we identified 2 synonymous single nucleotide polymorphisms (SNP) at positions 1179 (A/G) and 1296 (A/G) using the pooled DNA sequencing approach. The DNA was extracted from 28 Holstein sires and their 1,362 sons obtained from the Cooperative Dairy DNA Repository and from 913 cows from the University of Wisconsin resource population. Single nucleotide polymorphism 1296 was associated with a significant increase in productive life in both populations. This finding is similar to results previously obtained for the protease inhibitor gene, which is near UTMP and was also found to be associated with productive life in dairy cattle. Differential allelic expression of UTMP was observed in reproductive tissues obtained from 9 heterozygous individuals. The differential allelic expression observed in this study is consistent with other studies showing a correlation between allelic variation in gene expression and phenotypic variability.

Association of the OLR1 gene with milk composition in Holstein dairy cattle.

Oxidized low-density lipoprotein receptor (OLR1) is the major protein that binds, internalizes, and degrades oxidized low-density lipoprotein. The role of OLR1 in lipid metabolism and the results of previous whole-genome scan studies prompted the investigation of OLR1 as a candidate gene affecting milk composition traits. Direct cDNA and genomic sequencing of OLR1 revealed 2 single nucleotide polymorphisms (SNP) in exon 4, 5 SNP in intron 4, and 1 in the 3' untranslated region (UTR). Four intragenic haplotypes comprising SNP positions 7,160, 7,161, 7,278, 7,381, 7,409, 7,438, 7,512, and 8,232 were inferred. Haplotype analysis showed that one of the haplotypes was associated with a significant increase in fat yield and fat percentage. Single SNP analysis showed that allele C of SNP 8,232 (in the 3'-UTR) had significant effects on fat yield and fat percentage, whereas SNP 7,160 and 7,161 (in exon 4) had no significant effects. Both single SNP and haplotype analyses indicate that SNP 8,232 in the 3'-UTR is associated with milk fat yield and percentage and it may be in linkage disequilibrium with the functional polymorphism. To provide support for the hypothesis that SNP 8,232 is responsible for OLR1 expression, OLR1 expression levels in individuals bearing different genotypes were assessed. It was found that OLR1 expression was reduced in genotype AA individuals compared with CC and AC individuals, suggesting that A at position 8,232 may be the nucleotide causing decreased OLR1 expression. The 3'-UTR polymorphism found in this study might control translation or stability of OLR1 mRNA.

Changes in conception rate, calving performance, and calf health and survival from the use of crossbred Jersey x Holstein sires as mates for Holstein dams.

Differences in conception rates in matings of Holstein sires or F1 Jersey x Holstein sires to Holstein dams in the University of Wisconsin-Madison experimental herd were evaluated, as were differences in birth weight, dystocia, serum protein, serum IgG, fecal consistency, respiratory disease, and perinatal and pre-weaning mortality among the resulting calves. When mated to randomly chosen, lactating Holstein cows, Holstein sires (n = 74) and crossbred sires (n = 7) did not differ in male fertility. Calves from Holstein sires and multiparous Holstein dams (n = 99) were 1.9 kg heavier than calves from crossbred sires and multiparous Holstein dams (n = 211), leading to greater likelihood (odds ratio of 1.24) of dystocia. Furthermore, calves from crossbred sires and multiparous Holstein dams had higher serum protein and serum IgG levels between 24 and 72 h of age, as well as lower rates of perinatal and preweaning morality than calves from Holstein sires and multiparous or primiparous Holstein dams. Mean fecal consistency scores from birth to 7 d of age and number of days with scours also tended to be lower among calves from crossbred sires, compared with calves from Holstein sires. No differences were observed in the incidence or severity of respiratory disease. Results of this study suggest that introduction of Jersey genes via crossbreeding may lead to a reduction in dystocia and improvements in calf health and survival in Holstein herds. Future studies should address other traits related to dairy farm profitability, including milk composition, female fertility, longevity, feed efficiency, and resistance to infectious and metabolic diseases.

Effects of the osteopontin gene variants on milk production traits in dairy cattle.

Osteopontin (OPN) is a highly phosphorylated glycoprotein whose gene has been cloned and sequenced in different species. Several whole genome scans have identified quantitative trait loci (QTL) affecting milk production traits on bovine chromosome 6 close to the osteopontin gene (OPN) location. The presence of OPN in milk and its elevated expression in mammary gland epithelial cells together with previous QTL studies have prompted us to investigate the effects of OPN variants on milk production traits in the Holstein dairy cattle population. A single nucleotide polymorphism in intron 4 (C/T) was detected and primers were designed to amplify genomic DNA from 1362 bulls obtained from Cooperative Dairy DNA Repository and from 214 cows from the University of Wisconsin herd. For the Repository population, the C allele was associated with an increase in milk protein percentage and milk fat percentage. Correlation between milk protein percentage and milk fat percentage was about 0.57. For the University of Wisconsin herd, the estimates of the effects of allele C were in the same direction as for the Repository population, although these estimates did not reach statistical significance. Our results are consistent with other studies that showed a significant association of the microsatellite markers in the region of OPN with milk protein percentage and other correlated traits.

The fibroblast growth factor 2 gene is associated with embryonic mortality in cattle.

The objective of this study was to investigate the association of the fibroblast growth factor 2 (FGF2) gene with embryonic survival and fertilization rate in cattle. This gene was chosen because of its role in regulating trophectoderm expression of interferon-tau, the maternal pregnancy recognition factor in ruminants. To evaluate the effect of FGF2 on fertility traits, we produced in vitro-fertilized embryos from 281 Holstein cows and from 7 sires. A total of 4,542 in vitro fertilizations were performed, from which a total of 3,171 embryos were produced. Survival and fertilization rates were assessed at d 7 of embryonic development. Using the pooled DNA sequencing approach, we identified 2 SNP in FGF2, SNP11646 and SNP23. All sires and cows were genotyped for these SNP. For fertilization rate, no significant differences between genotypes were found for either SNP, whereas the effect on survival rate was significant for SNP11646. The survival rate of embryos produced from GG cows for this SNP was 37%, compared with 28 and 29% for embryos produced from AG and AA cows, respectively. Although the molecular mechanisms that cause embryonic mortality have not yet been identified, this study provides the first evidence of association between FGF2 and embryonic mortality in cattle. Thus, we propose that FGF2 can be used in animal breeding strategies to test for improved reproductive performance.

In vitro production of Holstein embryos using sex-sorted sperm and oocytes from selected cull cows.

The objective of this study was to explore potential synergies between sex-sorted sperm and in vitro embryo production for generating replacement heifers on commercial dairy farms. Selected involuntary cull cows (i.e., genetically suitable cows that were culled due to injury, illness, or infertility) from 7 Wisconsin farms were used as donors, and ovaries were collected via colpotomy or at the time of slaughter. Oocytes were aspirated, fertilized in vitro with sex-sorted sperm 22 +/- 0.2 h later, cultured, matured for 7 to 8 d, and transferred into recipient cows and heifers on the farms from which the cull cows originated. From August 2002 to June 2003, ovaries were recovered from 104 Holstein donors. Sex-sorted sperm from 3 Holstein sires (obtained via fluorescence-activated cell sorting) were used. A total of 365 transferable embryos were produced, an average of 3.6 +/- 0.3 per donor. However, due to limited availability of recipient animals, only 272 (fresh) embryos were transferred, an average of 2.6 +/- 0.3 per donor. A random subset of recipients received an injection (i.m.) of GnRH (100 microg) at the time of embryo transfer. When lactating cows were used as recipients, mean conception rates were 16.3% for recipients identified based on standing estrus and 20.0% for recipients synchronized using a timed breeding program (Ovsynch). Conception rates for in vitro-produced embryos were lower than corresponding conception rates for control cows inseminated using unsorted semen. When virgin heifers were used as embryo recipients (all standing estrus), the mean conception rate was 34.2%. The following effects significantly impacted conception rate: farm, season, recipient group (cow vs. heifer), sire of embryo, and GnRH injection. Of 40 full-term calves generated using sex-sorted semen, 37 were female. These results suggest that "low-cost" in vitro embryo production using cull cows as donors, in conjunction with sex-sorted sperm, could be an effective tool in dairy cattle breeding programs, but only if conception rates can be improved.

Inhibition of protein kinases after an induced calcium transient causes transition of bovine oocytes to embryonic cycles without meiotic completion.

We have examined the response of bovine oocytes matured in vitro for 24 hr to parthenogenic activation using compounds that increase intracellular calcium (ionomycin) or inhibit protein phosphorylation (6-dimethylaminopurine, DMAP). Treatment with ionomycin alone caused resumption of meiosis (57.8 +/- 7.8%) but not pronuclear formation (8.9 +/- 7.3%). DMAP alone did not cause resumption of meiosis or pronuclear formation. Sequential treatment with ionomycin (5 microM for 4 min) immediately followed by DMAP (1.9 mM for 5 hr) resulted in activation that led to pronuclear formation (80.5 +/- 13.1%). Completion of meiosis, however, was bypassed as evidenced by only one polar body and one pronucleus present in activated parthenogenones. It was necessary to incubate the oocytes for at least 3 hr in DMAP to obtain high rates of activation (76.6 +/- 9.8%) and development to blastocysts (21.1 +/- 1.5%). Temporal separation of the two treatments resulted in a decrease in oocytes with one pronucleus and one polar body (uniformly diploid parthenogenones) and an increase in a mixture of diploid and haploid parthenogenones since DMAP was capable of causing transition to interphase of all chromatin configurations after anaphase commenced and prior to metaphase arrest. Parthenotes produced with ionomycin and DMAP that developed to the blastocyst stage had high cell numbers (70 to 88 cells) and were able to cause extended cycles in 33.3% of recipient cattle after nonsurgical transfer to the uterus. Response of the bovine oocyte arrested in metaphase II to different activation stimuli was also found to show age-dependent changes in pattern of activation response and developmental competence.