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Background Methane emission by ruminants has contributed considerably to the global warming and understanding the genomic architecture of methane production may help livestock producers to reduce the methane emission from the livestock production system. The goal of our study was to identify genomic regions affecting the predicted methane emission (PME) from volatile fatty acids (VFAs) indicators and VFA traits using imputed whole-genome sequence data in Iranian Holstein cattle. Results Based on the significant-association threshold (p − 8), 33 single nucleotide polymorphisms (SNPs) were detected for PME per kg milk (n = 2), PME per kg fat (n = 14), and valeric acid (n = 17). Besides, 69 genes were identified for valeric acid (n = 18), PME per kg milk (n = 4) and PME per kg fat (n = 47) that were located within 1 Mb of significant SNPs. Based on the gene ontology (GO) term analysis, six promising candidate genes were significantly clustered in organelle organization (GO:0004984, p = 3.9 × 10− 2) for valeric acid, and 17 candidate genes significantly clustered in olfactory receptors activity (GO:0004984, p = 4 × 10− 10) for PME traits. Annotation results revealed 31 quantitative trait loci (QTLs) for milk yield and its components, body weight, and residual feed intake within 1 Mb of significant SNPs. Conclusions Our results identified 33 SNPs associated with PME and valeric acid traits, as well as 17 olfactory receptors activity genes for PME traits related to feed intake and preference. Identified SNPs were close to 31 QTLs for milk yield and its components, body weight, and residual feed intake traits. In addition, these traits had high correlations with PME trait. Overall, our findings suggest that marker-assisted and genomic selection could be used to improve the difficult and expensive-to-measure phenotypes such as PME. Moreover, prediction of methane emission by VFA indicators could be useful for increasing the size of reference population required in genome-wide association studies and genomic selection.

Many studies have been shown that freezing induced oxidative stress has detrimental effect on post-thaw sperm quality.This study was conducted to investigate the effect of tert-butyl hydroquinone (tBHQ) on bull semen crtopreservation.In this study, four different levels of tBHQ [Optidyl containing zero (T0), 2.5 (T2.5), 5 (T5), and 7.5 µM (T7.5) tBHQ] was used to study the effect of tBHQ on freezability of bull semen. On each collection day, four ejaculates were collected (a total of 24 ejaculates from four bulls), pooled and divided to four equal parts. Each part was diluted with one of the above-mentioned extenders and frozen. After thawing, sperm motility, plasma membrane functionality and integrity, apoptosis status and mitochondrial activity were assessed.The results show that total sperm motility was significantly higher in T5 compared to other groups. The value of VSL was significantly lower in T5 compared to T0. Also, T5 resulted in lower LIN and STR versus T0 and T2.5 groups. All extenders containing tBHQ resulted in a significantly higher percentage of sperm with functional membrane compared to T0 groups. Finally, Apoptosis related parameters and mitochondrial activity were not significantly difference between the groups.adding 5 µM tBHQ to the bull semen extender can be beneficial for post-thaw sperm quality. Also, in vivo or in vitro fertility test is recommended to test fertilizing ability of tBHQ exposed sperm.

There is a growing interest in odd- and branched-chain fatty acids (OBCFA) in milk following reports that several branched-chain fatty acids (FA) have health promoting effects, and certain milk OBCFA could serve as a biomarker to assess ruminal function. Twenty-four Holstein cows were fed 3 low-forage diets containing 30 g/kg of dry matter of prilled palm fat (PPF), sunflower oil (SO), or an equal mixture of both fats (experiment 1) or 3 diets containing 30 g/kg of dry matter of SO with a forage-to-concentrate ratio of 39:61, 44:56, or 48:52 (Experiment 2); diets were fed to investigate milk OBCFA composition and to explore the relationships between ruminal VFA and milk OBCFA using principal component analysis. Including SO in diets decreased yields of milk 13:0 anteiso, 15:0 anteiso, 15:0, 17:0, cis-9 15:1, and cis-9 17:1 compared with PPF. The molar proportion of ruminal propionate was the lowest and the yields of milk 14:0 iso and 16:0 iso were the greatest with the diet containing both fat supplements. Replacing concentrate with forages linearly increased ruminal acetate and yields of milk 13:0 iso, 14:0 iso, 15:0 iso, 16:0 iso, 17:0 iso, 13:0 anteiso, 15:0 anteiso, 15:0, 17:0, cis-9 15:1, and cis-9 17:1. The principal component analysis revealed that ruminal molar proportion of acetate related to concentrations of milk iso FA containing

This study was performed to investigate the effect of sub-lethal exposure of bull semen to ethanol on the post-thaw spermatozoa quality. Semen samples (n=24, 6 ejaculates/bull) from 4 Holstein bulls were collected and pooled. Pooled samples were divided into 4 equal parts and each part was frozen after being diluted with Optidyl® extender containing 0 (O-E0), 0.03 (O-E3), 0.09 (O-E9) and 0.15 (O-E15) % (v/v) absolute ethanol. Sperm motility and velocity, plasma membrane integrity and functionality, mitochondrial activity, malondialdehyde concentration, and apoptosis status were evaluated after thawing. A higher percentage of total motility was observed in the O-E9 group as compared to the O-E0, O-E3 and O-E15 groups (p0.05). Also, plasma membrane integrity was higher (p0.05) in the O-E9 group compared to the O-E3, and O-E15 groups. However, the difference between the O-E9 and O-E0 groups was not significant (p0.05). In terms of the proportion of sperm abnormality and plasma membrane functionality no differences (p0.05) were observed between the groups. Our results revealed that malondialdehyde level was lower in ethanol treated (O-E3, O-E9 and O-E15) groups compared to the O-E0 group (p0.05). Furthermore, the percentage of live spermatozoa with active mitochondria was higher in the O-E9 and O-E15 groups compared to the O-E0 and O-E3 groups (p0.05). The O-E3 and O-E9 groups resulted in the highest and lowest percentage of apoptotic spermatozoa, respectively (p0.05). The results of this study demonstrate that supplementation of semen extender with sub-lethal concentration of ethanol influences post-thawed bull sperm quality in a dose dependent manner.

Background Genome sequencing and subsequent gene annotation of genomes has led to the elucidation of many genes, but in vertebrates the actual number of protein coding genes are very consistent across species (~20,000). Seven years after sequencing the cattle genome, there are still genes that have limited annotation and the function of many genes are still not understood, or partly understood at best. Based on the assumption that genes with similar patterns of expression across a vast array of tissues and experimental conditions are likely to encode proteins with related functions or participate within a given pathway, we constructed a genome-wide Cattle Gene Co-expression Network (CGCN) using 72 microarray datasets that contained a total of 1470 Affymetrix Genechip Bovine Genome Arrays that were retrieved from either NCBI GEO or EBI ArrayExpress. Results The total of 16,607 probe sets, which represented 11,397 genes, with unique Entrez ID were consolidated into 32 co-expression modules that contained between 29 and 2569 probe sets. All of the identified modules showed strong functional enrichment for gene ontology (GO) terms and Reactome pathways. For example, modules with important biological functions such as response to virus, response to bacteria, energy metabolism, cell signaling and cell cycle have been identified. Moreover, gene co-expression networks using “guilt-by-association” principle have been used to predict the potential function of 132 genes with no functional annotation. Four unknown Hub genes were identified in modules highly enriched for GO terms related to leukocyte activation (LOC509513), RNA processing (LOC100848208), nucleic acid metabolic process (LOC100850151) and organic-acid metabolic process (MGC137211). Such highly connected genes should be investigated more closely as they likely to have key regulatory roles. Conclusions We have demonstrated that the CGCN and its corresponding regulons provides rich information for experimental biologists to design experiments, interpret experimental results, and develop novel hypothesis on gene function in this poorly annotated genome. The network is publicly accessible at http://www.animalgenome.org/cgi-bin/host/reecylab/d. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3176-2) contains supplementary material, which is available to authorized users.

Background Weighted Gene Co-expression Network analysis, a powerful technique used to extract co-expressed gene pattern from mRNA expression data, was constructed to infer common immune strategies used by cattle in response to five different bacterial species (Escherichia coli, Mycobacterium avium, Mycobacterium bovis, Salmonella and Staphylococcus aureus) and a protozoa (Trypanosoma Congolense) using 604 publicly available gene expression microarrays from 12 cattle infection experiments. Results A total of 14,999 transcripts that were differentially expressed (DE) in at least three different infection experiments were consolidated into 15 modules that contained between 43 and 4441 transcripts. The high number of shared DE transcripts between the different types of infections indicated that there were potentially common immune strategies used in response to these infections. The number of transcripts in the identified modules varied in response to different infections. Fourteen modules showed a strong functional enrichment for specific GO/pathway terms related to “immune system process” (71%), “metabolic process” (71%), “growth and developmental process” (64%) and “signaling pathways” (50%), which demonstrated the close interconnection between these biological pathways in response to different infections. The largest module in the network had several over-represented GO/pathway terms related to different aspects of lipid metabolism and genes in this module were down-regulated for the most part during various infections. Significant negative correlations between this module’s eigengene values, three immune related modules in the network, and close interconnection between their hub genes, might indicate the potential co-regulation of these modules during different infections in bovine. In addition, the potential function of 93 genes with no functional annotation was inferred based on neighbor analysis and functional uniformity among associated genes. Several hypothetical genes were differentially expressed during experimental infections, which might indicate their important role in cattle response to different infections. Conclusions We identified several biological pathways involved in immune response to different infections in cattle. These findings provide rich information for experimental biologists to design experiments, interpret experimental results, and develop novel hypothesis on immune response to different infections in cattle. Electronic supplementary material The online version of this article (10.1186/s12865-017-0238-4) contains supplementary material, which is available to authorized users.

Embryo transfer is a reproductive technique that has a major impact on the dissemination of economically important genes and the rate of genetic gain in breeding schemes. In recent years, there has been increasing interest in the use of sexed and genotyped embryos in commercial embryo transfer programs. Marker/gene assisted selection (MAS/GAS) projects can be performed in the pre-implantation stage through mass production of characterized embryos. Biopsy of a few cells in the morulla stage is essential for pre-implantation genetic diagnosis (PGD), in which sex determination, evaluation of disease genes, and genotyping for candidate genes are performed. Limited quantity of cells and low amount of DNA restrict the use of multiple molecular analyses in PGD programs. Recently, whole genome amplification (WGA) techniques promise to overcome this problem by providing sufficient input DNA for analysis. Among several techniques proposed for WGA, the primer extension pre-amplification (PEP) and the improved-primer extension pre-amplification (I-PEP) methods are the most commonly used. However, these methods are time-consuming and need more than 12 h amplification cycles. Since the time is a critical parameter in the successful characterized embryo transfer, the shortening of diagnosis time is highly desirable. In this study, we developed a short and simple I-PEP procedure (~3 h) and evaluated its performance for the amplification of bovine genomic DNA. We assessed short WGA procedure by polymerase chain reaction (PCR) amplification of 7 specific loci. The results indicated that the short procedure possesses enough sensitivity for the molecular genetic analysis of 1 input cell. Although the efficiency of the method was 100%, there was an inconsistency between genomic DNA (gDNA) and whole genome amplification product (wgaDNA) genotypes for kappa-casein locus; that is, however, most likely due to allele drop-out (ADO) or false homozigocity. The results of this study indicate that with the application of reliable methods, WGA-amplified bovine DNA will be a useful source for sexing and genotyping bovine embryos in several quantitative trait locus (QTL) markers.

A stochastic bio-economic model was used to determine the effects of applying artificial insemination (AI) with conventional or sexed semen at different conception rates and herd sizes on the genetic progress of productive trait and net profit over a 15-year period. The genetic means of the cattle population was predicted to increase over the years and the rate of genetic progress in the population program using sexed semen was significantly greater than the rate of progress in the population using conventional semen. The theoretical genetic gain was greater than the realized genetic gain for milk yield for all the AI scenarios. In addition, the genetic means for milk yield in the expanding herds were greater than in the fixed sized herds. The trend in net profit generated by using AI strategies with conventional semen over the time horizon was greater when herds were expanding (P < 0.01). The trend in net profit for fixed sized herds was generally negative, however, expanding herds experienced positive net profits over the years. Also, the net profit of using AI strategies with sexed semen was greater when dairy herds were expanding. On the other hand, the net profit of using AI with sexed semen was greater than that of AI with conventional semen at the end of the time horizon. The results of sensitivity analyses indicated that a 20% increase or decrease in milk sales income and feed costs have significantly modified the net profit of AI strategies using either sexed or conventional semen.

The main objective of this study was to estimate the genetic and phenotypic relationships between calving difficulty (CD) and fertility traits, including success at first service (SF), number of inseminations to conception (INS), interval from calving to first service (CTFS), interval between first and last service (IFL) and days open (DO), in first-parity Iranian Holsteins under standard (SMMs) and recursive (RMMs) mixed models. The data analysed in this paper included 29 950 records on CD and fertility traits, collected in the time period from 1995 to 2014 by the Animal Breeding and Improvement Center of Iran. Under all observed SMMs and RMMs, five bivariate sire-maternal grandsire models (ten bivariate analyses in total) were used for the analyses. Recursive models were applied with a view to consider that CD influences the fertility traits in the subsequent reproductive cycle and the genetic determination of CD and fertility traits by fitting CD as covariate for any of the fertility traits studied. The existence of such cause-and-effect is considered in RMMs but not in SMMs. Our results implied a statistically non-zero magnitude of the causal relationships between CD and all the fertility traits studied, with the former influencing the latter. The causal effects of CD on SF (on the observed scale, %), INS, CTFS, IFL and DO were -2.23%, 0.10 services, 1.93 days, 3.76 days and 5.61 days, respectively. Direct genetic correlations between CD and the fertility traits under both models were not statistically different from zero (95% HPD interval included zero), except for the correlation between CD and CTFS, which were 0.197 and 0.134 under SMM and RMM, respectively, indicating that genes associated with difficult births also increase intervals between calving and the first insemination afterwards. Comparison of both models by the deviance information criterion (DIC) demonstrated the plausibility of RMMs over SMMs.

Different fertility indices were constructed for improving fertility performance in Iranian Holstein dairy cows. Number of inseminations per conception and days from calving to first insemination, each weighted by its economic value, were included as breeding goals in the aggregate genotype definition. Different fertility indices (FI) were constructed with different combinations of available fertility traits number of inseminations to conception (INS), days from calving to first service (DFS), interval between first and last insemination (IFL), and days open (DO). The fertility index (FI1) that included INS and DFS had the greatest genetic gain for INS (−0.39 insemination), DFS (−7.47 days), and profit ($4.3) per generation. Genetic gain for profit, DFS, and INS including only DO showed slight differences regarding FI1. A selection index that included only INS (DFS) presented the larger (smaller) genetic gains for INS and smaller (larger) for DFS, which were −0.40 (−0.034) and −0.975 (−11.18) inseminations and days, respectively. The result of this study showed that recording INS and DFS are preferable traits for including in a fertility subindex. DO can be used in the absence of other fertility traits. © 2014, Springer Science+Business Media Dordrecht.

On the basis of the potential benefits to human health there is an increased interest in producing milk containing lower-saturated fatty acid (SFA) and higher unsaturated fatty acid (FA) concentrations, including cis-9 18:1 and cis-9, trans-11-conjugated linoleic acid (CLA). Twenty-four multiparous Holstein cows were used in two experiments according to a completely randomized block design, with 21-day periods to examine the effects of incremental replacement of prilled palm fat (PALM) with sunflower oil (SFO) in high-concentrate diets containing 30 g/kg dry matter (DM) of supplemental fat (Experiment 1) or increases in the forage-to-concentrate (F : C) ratio from 39 : 61 to 48 : 52 of diets containing 30 g/kg DM of SFO (Experiment 2) on milk production, digestibility and milk FA composition. Replacing PALM with SFO had no effect on DM intake, but tended to increase organic matter digestibility, yields of milk, protein and lactose, and decreased linearly milk fat content. Substituting SFO for PALM decreased linearly milk fat 8:0 to 16:0 and cis-9 16:1, and increased linearly 18:0, cis-9 18:1, trans-18:1 (��4 to 16), 18:2 and CLA concentrations. Increases in the F : C ratio of diets containing SFO had no effect on intake, yields of milk, milk protein or milk lactose, lowered milk protein content in a quadratic manner, and increased linearly NDF digestion and milk fat secretion. Replacing concentrates with forages in diets containing SFO increased milk fat 4:0 to 10:0 concentrations in a linear or quadratic manner, decreased linearly cis-9 16:1, trans-6 to -10 18:1, 18:2n-6, trans-7, cis-9 CLA, trans-9, cis-11 CLA and trans-10, cis-12 CLA, without altering milk fat 14:0 to 16:0, trans-11 18:1, cis-9, trans-11 CLA or 18:3n-3 concentrations. In conclusion, replacing prilled palm fat on with SFO in high-concentrate diets had no adverse effects on intake or milk production, other than decreasing milk fat content, but lowered milk fat medium-chain SFA and increased trans FA and polyunsaturated FA concentrations. Increases in the proportion of forage in diets containing SFO increased milk fat synthesis, elevated short-chain SFA and lowered trans FA concentrations, without altering milk polyunsaturated FA content. Changes in fat yield on high-concentrate diets containing SFO varied between experiments and individual animals, with decreases in milk fat secretion being associated with increases in milk fat trans-10 18:1, trans-10, cis-12 CLA and trans-9, cis-11 CLA concentrations.

Stochastic simulation was used for studying the impacts of sexed semen on genetic progress and reproductive performance of dairy cows. Three strategies were compared: WSS (use unsexed semen in cows and heifers), SSH (use sexed semen in heifers and unsexed semen in cows) and SSCH (use sexed semen in both cows and heifers). Conception rate (CR) of unsexed semen was considered to be 35% and 65% in cows and heifers, respectively. CR of sexed semen was considered to be 15 (20% in cows and 50% in heifers), 10, 5 and 0 percentage points lower than unsexed semen. Thus, four subschemes were compared under SSCH (SSCH15, SSCH10, SSCH5, SSCH0) and SSH (SSH15, SSH10, SSH5, SSH0). Moreover, the effect was studied in four distinct paths of selection: active sires (AS), young bulls (YB), bull dams (BD) and milking cows (CW). The average genetic superiority of CW was 12% and 9.5% in SSCH15 and SSH15 strategies relative to a base scheme, respectively. The average genetic superiority of CW was 19% and 10.5% in SSCH0 and SSH0, respectively. Regression analysis showed that genetic superiority of CW increased significantly, that is, 0.5% and 0.1% per every 1% increase in CR in SSCH and SSH, respectively. The result showed that there is a significant difference between genetic superiority of cows in SSCH and SSH schemes. Widespread and limited use of sexed semen in commercial dairy herds resulted in a large genetic advantage in CW. The genetic advantage of gender control was minimal in the selection paths of AS, YB and BD. Open days and services per conception reached to 153 v. 125 days and 5 v. 2.86 under SSCH15 compared with WSS. The age at first calving increased from 774 to 790 days in SSH15 and SSCH15 strategies. Mean of parities decreased to 2.26 v. 2.42 by using sexed semen. The widespread use of sexed semen increased the age average of cows in all parities. Sexed semen increased selection intensity in the CW path, and this contributed to the genetic merit of future cows. On the other hand, sexed semen had a negative effect on the reproductive performance of dairy cows. Generally, although the effect of widespread use of sexed semen (SSCH) on genetic progress is significantly more than limited use of sexed semen (SSH), SSCH decreased reproductive performance of dairy herds dramatically, and this suggests that SSH scenarios might be more appropriate in animal breeding programs. Finally, to make a decision of which schemes are more convenient, it is necessary to compare the economic aspects of schemes.

Trait-by-trait and multiple trait bioeconomic modeling were used to derive farm-specific economic weights (EW) for a wide range of traits under different production and economic circumstances to define breeding objectives for Holstein dairy cattle in Iran. Production parameters and economic data were gathered on 10 dairy farms from March 2008 to February 2010. The EW (economic values multiplied by gene expressions, in US dollars per unit of trait per calf born from sires of self-replacing females in planning horizon of 20 yr) were estimated to be $0.15 per kilogram of milk yield; $1.36 per kilogram of fat yield; −$1.02 per kilogram of protein yield; $4.59 per month of longevity; −$1.22 per kilogram of mature cow weight; −$105.67 for combined somatic cell score and clinical mastitis; −$1.35 and −$0.28 for percentage direct and maternal calving difficulties, respectively; −$3.98 for percentage direct stillbirth; −$0.76 per day of age at first calving; −$0.72 per calving interval day; and $0.91 for percentage 56-d nonreturn rate on averages across investigated farms. The coefficient of variation of economic weights across the 10 farms was lowest for direct calving difficulty and highest for calving interval. The proposed Iranian selection index was compared with selection indices of major countries exporting semen to Iran. Average relative emphasis for production, durability, and health and reproduction, across all exporter countries, was 41, 37.5, and 21.5%, respectively, whereas the respective values were 50, 14, and 36% for the Iranian index. Significant differences in selection indices may potentially decrease the utility of importation of semen as a means of achieving sustainable genetic progress in Iran. Results obtained in this study provide important information about economic values of traits that can be used to improve the Iranian national progeny testing program as well as importation rules for semen to Iran.

Calving records from the Animal Breeding Center of Iran, collected from January 1991 to December 2007 and comprising 1,163,594 Holstein calving events from 2,552 herds, were analyzed using a linear animal model, linear sire model, threshold animal model, and threshold sire model to estimate variance components, heritabilities, genetic correlations, and genetic trends for twinning rate in the first, second, and third parities. The overall twinning rate was 3.01%. Mean incidence of twins increased from first to fourth and later parities: 1.10, 3.20, 4.22, and 4.50%, respectively. For first-parity cows, a maximum frequency of twinning was observed from January through April (1.36%), and second- and third-parity cows showed peaks from July to September (at 3.35 and 4.55%, respectively). The phenotypic rate of twinning decreased from 1991 to 2007 for the first, second, and third parities. Sire predicted transmitting abilities were estimated using linear sire model and threshold sire model analyses. Sire transmitting abilities for twinning rate in the first, second, and third parities ranged from -0.30 to 0.42, -0.32 to 0.31, and -0.27 to 0.30, respectively. Heritability estimates of twinning rate for parities 1, 2, and 3 ranged from 1.66 to 10.6%, 1.35 to 9.0%, and 1.10 to 7.3%, respectively, using different models for analysis. Heritability estimates for twinning rate, obtained from the analysis of threshold models, were greater than the estimates of linear models. Solutions for age at calving for the first, second, and third parities demonstrated that cows older at calving were more likely to have twins. Genetic correlations for twinning rate between parities 2 and 3 were greater than correlations between parities 1 and 2 and between parities 1 and 3. There was a slightly increasing trend for twinning rate in parities 1, 2, and 3 over time with the analysis of linear animal and linear sire models, but the trend for twinning rate in parities 1, 2, and 3 with threshold animal model analysis was decreased over the years. There was a significant decreasing trend for twinning rate in parities 1 and 2 over time with the threshold sire model analysis, but the genetic trend for twinning rate in parity 3 with this model of analysis was significant and positive. In general, there were increasing genetic trends for twinning rate from parities 1 through 3 using different models of analysis.

The aim of this study was to estimate the allele frequencies in polymorphic site of exon six of POU1F1 gene in three Iranian native and Holstein cattle. Genomic DNA was extracted from 3 Iranian native cattle breeds, including 97 Mazandarani, 87 Sarabi, 112 Golpaygani and also 110 Holstein cattle. A 451 bp fragment of intron 5 and exon 6 were amplified and digested with HinfI restriction enzyme. Frequencies of allele A were 0.37, 0.27, 0.34 and 0.21 for Mazandarani, Sarabi, Golpaygani and Holstein cattle, respectively. Significant differences in genotype frequencies were found between Mazandarani or Golpaygani and Holstein cattle. No significant differences in genotype frequencies were found between Sarabi and Holstein cattle. Transition A to G in nucleotide 1256 is responsible for HinfI(-) allele. No significant association was observed between POU1F1 polymorphism and milk production. Differences in allelic frequency between native Bos indicus breeds (Mazandarani, Golpaygani) and Holstein at the present study might be due to differences in origin breeds, low number of samples and/or as the effect of natural selection in native breeds.

Calving records of Holstein dairy cows from April 1998 to September 2006 comprising 16 herds with 104,572 calving events representing 4,045 twin births were used to evaluate reported twinning rate, calf sex ratio, stillbirth, and abortion rates in single and twin births. Overall, the reported twinning rate was 3.9%, and twinning increased with parity [1.1% for primiparous cows vs. 5.7% for cows in their fourth or greater lactation; odds ratio (OR) = 5.50]. Regardless of parity, the greatest twinning rate was observed when conception occurred in fall season from September to December (OR = 1.17). Calf stillbirth was greater after twin births, with 18.8% of twin calving events resulting in one or both calves as stillborn, compared with 4.0% for singleton births (OR = 7.58). Calf stillbirth for multiparous cows was 2.9% for single births and 18.0% for twin births (OR = 7.08), whereas for primiparous cows, stillbirth was 6.6% for singletons and 27.7% for twins (OR = 5.85). Calf sex ratios (male, M; female, F) were 52.4% M and 47.6% F for singleton calves and 28.2% MM, 48.9% MF, and 22.9% FF for twin calves. The mean abortion rate was 13.4%, with 13.8% for single births and 4.2% for twin births (OR = 1.22). Abortion rate for multiparous cows was 15.9% for single births and 4.0% for twin births (OR = 4.31), whereas for primiparous cows, abortion rate was 9.4% for single births and 5.4% for twin births (OR = 1.89). Although milk production, as a causative factor associated with twinning, increased in recent years, twinning rate decreased over the years.