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Background Maternal nutrition during gestation affects fetal development, which has long-term programming effects on offspring postnatal growth performance. With a critical role in protein and lipid metabolism, essential fatty acids can influence the development of muscle and adipose tissue. The experiment investigated the effects of late gestation supplements (77 d prepartum), either rich in saturated and monounsaturated fatty acids (CON; 155 g/cow/d EnerGII) or polyunsaturated fatty acids (PUFA; 80 g/cow/d Strata and 80 g/cow/d Prequel), on cow performance and subsequent calf growth performance as well as mRNA expression in longissimus muscle (LM) and subcutaneous adipose tissue at birth and weaning. Results There was no difference (P ≥ 0.34) in cow body weight (BW) or body condition score from pre-supplementation through weaning. Relative concentrations of C18:3n-3 and C20:4n-6 decreased (P ≤ 0.05) to a greater extent from mid-supplementation to calving for PUFA compared with CON cows. Cow plasma C20:0, C20:5n-3, and C22:6n-3 were increased (P ≤ 0.01) in PUFA during supplementation period. At birth, PUFA steers had greater (P = 0.01) plasma C20:5n-3. No differences (P ≥ 0.33) were detected in steer birth BW or dam milk production, however, CON steers tended (P = 0.06) to have greater pre-weaning average daily gain and had greater (P = 0.05) weaning BW compared with PUFA. For mRNA expression in steers: MYH7 and C/EBPβ in LM increased (P ≤ 0.04) to a greater extent from birth to weaning for PUFA compared with CON; MYF5 in LM and C/EBPβ in adipose tissue tended (P ≤ 0.08) to decrease more from birth to weaning for CON compared with PUFA; SCD in PUFA adipose tissue tended (P = 0.08) to decrease to a greater extent from birth to weaning than CON. In addition, maternal PUFA supplementation tended (P = 0.08) to decrease MYOG mRNA expression in LM and decreased (P = 0.02) ZFP423 in adipose tissue during the pre-weaning stage. Conclusions Late gestation PUFA supplementation decreased pre-weaning growth performance of the subsequent steer progeny compared with CON supplementation, which could have been a result of downregulated mRNA expression of myogenic genes during pre-weaning period.

Mature Simmental × Angus cows [n = 147; body weight (BW) = 590 ± 72 kg] were used to evaluate the effects of early weaning on subsequent parity calf growth performance and carcass characteristics. Cows were assigned to one of two treatments based on their previous calf’s weaning age: early wean (EW) or conventional wean (CW). Dams assigned to the EW treatment had calves previously weaned at 88 ± 6 d of age, whereas dams assigned to the CW treatment had calves previously weaned at 185 ± 6 d of age. Cow BW and body condition scores (BCS) were monitored during the experiment. All cows were managed as a common group from the onset of the experiment at breeding until final pregnancy check of their next production cycle 462 d later. All calves in the experiment were managed as one group and weaned at a single time point, then feedlot performance and carcass characteristics were evaluated. Initial cow BW was different (P < 0.05), so it was included as a covariate for cow BW analysis. There was a treatment × date interaction (P < 0.01) for cow BW and cow BCS. Cow BW was consistently greater for the EW treatment from day 39 to the end of the experiment (P < 0.01). Cow BCS were not different at the onset of the experiment (P = 0.20), although after breeding and throughout lactation, BCS diverged between treatments and the EW treatment consistently had greater (P < 0.01) BCS than the CW treatment throughout the entire subsequent lactation. Gestation length was not different (P = 0.21) between treatments, yet calf birth BW was greater (P = 0.05) for the EW treatment. Neither artificially inseminated pregnancy percentage nor overall pregnancy percentage was different between treatments (P ≥ 0.61). Despite the greater birth BW for the EW treatment and no difference (P = 0.25) in milk production, weaning BW was not different (P = 0.50) between treatments. Feedlot performance measures were not different (P ≥ 0.13) between treatments, including feedlot arrival weight, final BW, average daily gain, dry matter intake, and gain to feed. Final carcass characteristics were not different (P ≥ 0.33) between treatments, including dressing percentage, hot carcass weight, LM area, 12th rib fat thickness, kidney, pelvic, heart fat percentage, USDA yield grade, and marbling score. Thus, early weaning resulted in improved BW and BCS of cows as well as increased birth BW of subsequent calf, although that did not transpire into differences in postnatal growth performance or carcass traits.

Simple Summary Maternal nutrition during late gestation affects fetal muscle and adipose tissue development, leading to persistent impacts on offspring postnatal growth and production performance. Fatty acids, especially essential fatty acids, play important roles in regulating protein and lipid metabolism. Therefore, the objective of the current study was to investigate the effects of maternal fatty acid supplementation during late gestation on beef cattle progeny finishing phase growth performance, carcass characteristics, and mRNA expression of genes involved in muscle and adipose tissue development. This study demonstrated that maternal supplementation with polyunsaturated fatty acids during late gestation increased the offspring’s feed ratio during the finishing phase. However, it had limited effects on finishing phase body weights, carcass characteristics, or relative mRNA expression in longissimus muscle and subcutaneous adipose tissue. Therefore, operations under similar systems could expect limited effects of maternal fatty acid supplementation on the growth and production performance of the finishing steers. Abstract The objective was to investigate the effects of feeding late gestational beef cows supplements differing in fatty acid profile on steer progeny finishing phase growth performance, carcass characteristics, and relative mRNA expression of myogenic and adipogenic genes. Seventy Angus-cross steers (initial body weight [BW] 273 ± 34 kg) born from dams supplemented with either 155 g DM/d EnerGII (CON, rich in palmitic and oleic acids) or 80 g DM/d Strata + 80 g DM/d Prequel (PUFA, rich in linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid) for the last 77 ± 6 d prepartum were used. Longissimus muscle and subcutaneous adipose biopsies were collected to evaluate relative mRNA expression of genes related to myogenesis and adipogenesis. Steers were slaughtered at 423 ± 6 d of age. No treatment × time interaction or treatment effect (p ≥ 0.21) was detected for steer finishing phase BW, while steers from PUFA supplemented dams tended (p = 0.06) to have a greater gain to feed ratio (G:F). Neither carcass characteristics nor relative mRNA expression was different (p ≥ 0.11). In conclusion, late gestation PUFA supplementation tended to increase steer progeny finishing phase G:F, but had no effects on finishing phase BW, carcass characteristics, or relative mRNA expression during the finishing phase.

The aim of this study was to investigate the impact of dietary crude protein (CP) levels and calcium salts of long-chain fatty acids (CSFA) on the nutrient intake and digestibility, nitrogen deposition, rumen fermentation characteristic and microbiota, plasma biochemical indexes and fatty acid composition in the Holstein bulls. Eight Holstein bulls was used in a 4 × 4 Latin Square design with 2 × 2 factorial diets, including two levels of CP (133 or 112 g/kg dry matter), and with or without 2.32 g/kg CSFA. The high CP level diets increased the CP intake and the CP apparent digestibility and the urinary nitrogen excretion (P = 0.001). Dietary supplementation with CSFA promoted the apparent digestibility of organic matter (P = 0.012). The diets of CSFA and high CP level raised the rumen ammonia nitrogen concentration (P = 0.009), and enhanced the isovalerate and valerate concentrations, respectively. The high CP diets improved the abundance of Butyrivibrio fibrisolvens (P = 0.037) and Megasphaera elsdenii (P = 0.023), while the CSFA reduced the abundance of methanogens (P = 0.047). High CP increased urea concentration (P = 0.008). Dietary supplementation with CSFA increased the cholesterol and the low-density lipoprotein cholesterol concentration in the plasma, and the proportion of C16:0, C18:1n9c and Δ9 desaturase C18, while it reduced the ratio of C21:1 and C22:0. The low CP diets reduced the protein waste and environmental pollution in the final stage of fattening Holstein bulls. Further study needs to be done to investigate the effect of CSFA on the CH4 emission in terms of microbial mechanism in the rumen.

Background Maternal nutrition during gestation affects fetal development, which has long-term programming effects on offspring postnatal growth performance. However, limited research is available for fetal programming effects of fatty acid supplementation on beef cattle. With a critical role in protein and lipid metabolism, essential fatty acids can influence the development of muscle and adipose tissue. The experiment investigated the effects of late gestation supplements, either rich in saturated and monounsaturated fatty acids (CON) or polyunsaturated fatty acids (PUFA), on cow performance and subsequent calf growth performance as well as gene expression in longissimus muscle (LM) and subcutaneous adipose tissue. Results There was no difference (P ≥ 0.32) in cow body weight (BW) or body condition score from pre-supplementation through weaning. There were treatment × time interactions (P ≤ 0.05) for plasma C18:3 and C20:4 concentrations, in which C18:3 and C20:4 of both CON and PUFA cows were greater at mid-supplementation than those at calving, while CON cows at birth had the intermediate concentrations. Cow plasma C20:0, C20:5, and C22:6 were increased (P ≤ 0.01) in PUFA. At birth, PUFA steers had greater (P = 0.01) plasma C20:5 and lower (P P ≥ 0.45), while CON steers had greater (P = 0.05) weaning BW and tended (P = 0.06) to have greater pre-weaning average daily gain compared to PUFA. There were treatment × time interactions (P ≤ 0.02) for MYH7 and C/EBPβ expressions in LM; expression of MYH7 was greatest in CON at birth, while C/EBPβ was greatest in PUFA steers at weaning. There tended (P = 0.06) to be a treatment × time effect for MYF5, in which expression was the greatest in CON steers at birth. Maternal supplementation of PUFA tended (P ≤ 0.10) to decrease MYOG expression in LM and ZFP423 expression in adipose tissue during the pre-weaning stage. Conclusions Late gestation PUFA supplementation decreased pre-weaning growth performance of the subsequent steer progeny compared to CON supplementation, which could have been a result of downregulated expression of myogenic genes during pre-weaning period.

Objectives were to investigate the effects of supplementing Ca salts of fatty acids to beef cows during late gestation on the pre-weaning growth performance and gene expression of steer progeny. Ninety-six fall-calving, Angus x Simmental cows (BW = 601±76 kg) were randomly assigned into 8 pastures (4 pastures/treatment; 12 cows/pasture), supplemented soyhulls mixed with 80 g/cow/d Strata + 80 g/cow/d Prequel (PUFA, rich in linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid) or 155 g/cow/d EnerGII (SFA/MUFA, rich in palmitic and oleic acids) for 76.5±6.0 d prepartum. Cow/calf pairs were comingled after calving. Biopsies of Longissimus dorsi (LD) and subcutaneous adipose were taken at birth and 3 wk before weaning (186±6 d). The data were analyzed using the MIXED procedure of SAS. Supplementing PUFA increased (P < 0.01) cow plasma eicosapentaenoic acid and docosahexaenoic acid at midpoint of supplementation. At birth, PUFA steers had greater (P < 0.05) plasma EPA and lower (P < 0.01) plasma linolenic acid and oleic acid than SFA/MUFA steers. Weaning BW and pre-weaning ADG of the SFA/MUFA steers tended (P < 0.07) to be greater than PUFA steers. SFA/MUFA steers had increased (P = 0.04) expression of Zfp423 in adipose. There tended to be a treatment×time interaction (P = 0.06) for expression of Myf5 in LD; expression of Myf5 was greatest in SFA/MUFA steers at birth. There was a treatment×time interaction (P = 0.05) for expression of Pax7; expression was greater in SFA/MUFA steers at birth than any treatment at weaning. There were treatment×time interactions (P < 0.01) for expression of Myh7 and C/EBPβ in LD; expressions were greatest in PUFA steers at weaning. Supplementing Ca salts of PUFA to beef cows during late gestation altered plasma fatty acid profile of cow and calf, affected gene expression, and decreased offspring growth performance.

Objectives were to investigate the effects of late-gestation supplementation of Ca salts of fatty acids to beef cows on cow performance and offspring pre-weaning growth performance. One hundred and ninety fall-calving, Angus Simmental cows were ranked by BW, BCS, and age. Cows were randomly assigned into 12 endophyte-infected, tall fescue pastures (4 pastures/treatment; 15–16 cows/pasture). Cows were fed an isocaloric supplement with soybean hulls mixed with: whole-shelled corn (CON), 155 g/cow/d of EnerGII (SFA/MUFA), or 40 g/cow/d of Strata 120 g/cow/d of Prequel (PUFA) during the last 83 4.9 d of gestation. Cow BW and BCS were measured at the initial, middle point, and end of supplementation, as well as at breeding and weaning. Milk yield was evaluated by weigh-suckle-weigh technique at 675.0 d postpartum. Steers were weaned at 174 5.0 d of age. Growth performance, milk, and blood parameters were analyzed with MIXED and reproductive data were analyzed with GLIMMIX procedures of SAS. The BW and BCS of the cows were not different (P 0.19) from trial initiation through weaning. Birth BW of the steers was not different (P = 0.62). The AI or overall pregnancy rates were not different (P 0.88). There was no difference (P 0.12) in milk yield or components. However, C15:0 and total n-3 fatty acids in milk from dams supplemented with PUFA was greater (P 0.05) than CON, while SFA/MUFA was intermediate and not different than the others. There was no difference (P = 0.83) for weaning BW of the steers. In conclusion, late-gestation supplementation of Ca salts of PUFA had no effect on cow BW, BCS, or milk yield, but increased total n-3 fatty acids in milk. Neither birth BW nor weaning BW of steers were affected by maternal fatty acid supplementation.

Twenty-eight male, weaned Chinese Holstein calves ((156.8±33.4) kg) were used to investigate the effects of dietary forage to concentrate ratio (F:C) and forage length on nutrient digestibility, plasma metabolites, ruminal fermentation, and fecal microflora. Animals were randomly allocated to four treatments in a 2×2 factorial arrangement: whole-length forage (WL) with low F:C (50:50); WL with high F:C (65:35); short-length forage (SL) with high F:C (65:35); and SL with low F:C (50:50). Chinese wildrye was used as the only forage source in this trial. The grass in the SL treatments was chopped using a chaff cutter to achieve small particle size (∼50% particles >19 mm). Dry matter intake (DMI) and organic matter (OM) intake was increased by increasing both F:C (P0.05). Microflora similarity was 50–73% among the different treatments. It is concluded that the WL with high F:C (65:35) diet is suitable for weaned calves.

The purpose of this study was to assess the effects of calcium salt of long-chain fatty acids (CSFA) and alfalfa on beef cattle in the late fattening. 48 Holstein bulls were selected and randomly divided into 4 groups, feeding four dietary that Leymus chinensis with (LC) or with no (LN) 2.4% CSFA, and alfalfa replaced 50% Leymus chinensis with (AC) or with no (AN) 2.4% CSFA. The results indicated that alfalfa improved the feed conversion rate (P < 0.05). CSFA increased serum low density lipoprotein cholesterol, and reduced the cooking loss of Longissimus muscle (P < 0.05). CSFA and alfalfa reduced Acetate/Propionate. Alfalfa and CASF had significant additive effects on the apparent digestibility of dry matter, crude protein, neutral detergent fiber, acid detergent fiber, organic matter and rumen fermentation for acetate, isobutyrate, butyrate, isovalerate, total volatile fatty acids (P < 0.05). CSFA increased microbial diversity index when compared with alfalfa (P < 0.05), but no significant differences were detected in bacterial genera abundances among diets. The relative abundances of rumen bacterial genera have significant correlation with apparent digestibility of nutrients, rumen fermentation characteristics and serum biochemical parameters (P < 0.05). These results comprehensively evaluated the additive effects of alfalfa and CSFA on the application in Holstein bulls.

The objective was to investigate the effects of late-gestation supplementation of Ca salts of fatty acids to beef cows on the finishing phase growth performance and carcass characteristics of the steer progeny. Ninety-six multiparous, fall-calving, Angus × Simmental cows (BW = 601 ± 76 kg) were stratified by BW, BCS, and age and randomly allotted into 8 endophyte-infected tall fescue pastures with 12 cows/pasture. Cows were supplemented with soybean hulls mixed with either 155 g/cow/d of EnerGII (SFA/MUFA; enriched with palmitic and oleic acids) or 80 g/cow/d of Prequel + 80 g/cow/d of Strata (PUFA; enriched with linoleic acid, EPA, and DHA) for the last 77 ± 6 d of gestation. Steer progeny (n = 70) were weaned at 186 ± 6 d of age and transported to feedlot. Steers were fed receiving diets for 3 weeks and transitioned to finishing diets and adapted to GrowSafe feed bunks for individual daily feed intake measurement. Steers were weighed every 28 d during the finishing phase. Steers were slaughtered at commercial abattoir at 432 ± 6 d of age, and carcass data were obtained from USDA camera grading system. Data were analyzed using the MIXED procedure of SAS, and repeated measure was used for BW. There were no differences (P ≥ 0.13) for BW, ADG, DMI, or feed conversion during the finishing phase. There was no differences (P ≥ 0.22) observed in hot carcass weight, dressing percentage, 12th rib fat thickness, yield grade, ribeye area, marbling score, or KPH fat. In conclusion, maternal supplementation of Ca salts of fatty acids during late gestation did not affect steer progeny growth performance during the finishing phase or carcass characteristics.

OBJECTIVE This study investigated the effect of dietary crude protein (CP) supplementation on nutrient intake, nitrogen (N) utilisation, blood metabolites, ruminal fermentation and growth performance of young Holstein bulls. METHODS Twenty-one young bulls weighing 277±11.2 kg were equally divided into three groups and were offered diets formulated with low CP (LCP; 10.21% CP and 4.22% rumen degradable protein [RDP]), medium CP (MCP; 12.35% CP and 5.17% RDP) and high CP (HCP; 14.24% CP and 6.03% RDP). Yellow corn silage was used as a unique forage source and was mixed with concentrate. This mixed feed was given ad libitum to the young bulls included in the study. RESULTS Results showed that CP intake, blood urea nitrogen, N intake, total N excretion and N balance increased linearly with an increase in dietary CP level (p

The objective was to determine the effects of supplementing calcium salts of PUFA or SFA + MUFA during late gestation on performance of fall-calving beef cows through calving. Mature, Simmental x Angus cows confirmed pregnant to a synchronized artificial insemination (AI) and fetal sexed were selected prior to the start of the experiment. At 200 d of gestation, cows (n = 96; BW = 603 ± 72 kg) were stratified by age, AI service sire, fetal sex and BW and allotted to 8 groups. Groups were randomly assigned to 1 of 2 treatments (4 groups per treatment): SFA + MUFA (0.155 kg of EnerGII per cow per day on a DM basis) and PUFA (0.08 kg of Strata and 0.08 kg of Prequel per cow per day on a DM basis). The SFA + MUFA supplement included palmitic and oleic acid. The PUFA supplement included eicosapentaenoic acid, docosahexaenoic acid, and linoleic acid. All cows grazed endophyte-infected tall fescue and were supplemented 2 kg DM per cow per d of soybean hulls for 78 d during late gestation in addition to their treatment supplement. Body condition score and BW were collected at trial initiation, during supplementation, before calving, and after calving. Pen was the experimental unit and BW, BCS, and calf BW were analyzed using the MIXED procedure of SAS. The BW and BCS at trial initiation, during supplementation, before calving, and after calving of the cows supplemented PUFA were not different (P ≥ 0.38) than the BW and BCS of cows supplemented SFA + MUFA. Calf birth BW from cows supplemented with PUFA were not different (P = 0.87) than calf birth BW from cows supplemented with SFA + MUFA. Supplementing cows PUFA during late gestation in fall-calving cows did not affect BW, BCS or calf birth BW.

Background Freemartinism generally occurs in female offspring of dizygotic twins in a mixed-sex pregnancy. Most bovine heterosexual twin females are freemartins. However, about 10% of bovine heterosexual twin females are fertile. Farmers mostly cull bovine fertile heterosexual twin females due to the lack of a practical diagnostic approach. Culling of such animals results in economic and genetic-material losses both for dairy and beef industry. Methods In this study, a comparative test, including qualitative detection of SRY gene by polymerase chain reaction (PCR), quantitative detection of relative content of SRY by real-time quantitative polymerase chain reaction (qPCR), and quantitative detection of H-Y antigen, was performed to establish the most accurate diagnosis for freemartin. Twelve Holstein heterosexual twin females were used in this study, while three normal Holstein bulls and three normal Holstein cows were used as a positive and negative control, respectively. Results Polymerase chain reaction results revealed that SRY gene were absent in three heterosexual twin females and only two of them were verified as fertile in later age. The qPCR results showed that relative content of SRY was more than 14.2% in freemartins and below 0.41% in fertile heterosexual twin females. The H-Y antigen test showed no significant numerical difference between freemartin and fertile heterosexual twin female. Discussion Our results show that relative content of SRY quantified by qPCR is a better detection method for diagnosis of freemartin in Holstein cattle as compare to qualitative detection of SRY gene by PCR or quantitative detection of H-Y antigen. To the authors’ knowledge, this is the first time we applied qPCR to diagnosing freemartin by quantifying SRY gene and got relative SRY content of each freemartin and fertile heterosexual twin female. We concluded that low-level of SRY would not influence fertility of bovine heterosexual twin female.

This study presented the effects of alfalfa and calcium salts of long-chain fatty acids (CSFA) on feed intake, apparent digestibility, rumen fermentation, microbial community, plasma biochemical parameters, and fatty acid profile in Holstein freemartin heifers. Eight Holstein freemartin heifers were randomly divided into a 4 × 4 Latin Square experiment with 2 × 2 factorial diets, with or without alfalfa or CSFA. Dietary supplementation of CSFA significantly increased the apparent digestibility of dry matter, crude protein, neutral detergent fiber, organic matter, and significantly reduced N retention (P < 0.05). CSFA increased the concentration of ammonia nitrogen in the ruminal fluid (P < 0.05), but alfalfa increased the concentration of valerate and isovalerate (P < 0.05). CSFA increased the concentration of ammonia nitrogen and the relative population of Streptococcus bovis in the rumen (P < 0.05) and inhibited the relative population of Ruminococcus flavefaciens, methanogens, and protozoa (P < 0.05). Alfalfa instead of Leymus chinensis increased the relative population of Butyrivibrio fibrisolvens and Ruminobacter amylophilus in the rumen (P < 0.05) and reduced the relative population of the Ruminococcus albus and Megasphaera elsdenii (P < 0.05). Supplemental CSFA increased the concentration of cholesterol and low-density lipoprotein cholesterol in the plasma (P < 0.05). And it also altered the composition of fatty acids in the plasma, which was expressed in reducing saturated fatty acid (ΣSFA) ratio and C14-C17 fatty acids proportion except C16:0 (P < 0.05) and increasing the proportion of polyunsaturated fatty acid (ΣPUFA) and unsaturated fatty acid (ΣUFA) (P < 0.05). The results showed that alfalfa and CSFA had interaction effect on the apparent digestibility of ether extracts, plasma triglyceride concentration, isobutyrate concentration, and Ruminococcus albus relative abundance in the rumen. It was concluded that alfalfa substituting Leymus chinensis did not change the apparent digestibility of nutrients in the final stage of fattening Holstein freemartin heifers, while CSFA increased the cholesterol and the proportion of unsaturated fatty acids in plasma. Alfalfa and CSFA had mutual interaction effect on fat digestion and plasma triglycerides.

Twelve Holstein bulls were used in a 4 × 4 Latin square design to investigate the effects of using whole-crop wheat hay (WCWH) as a substitute for Leymus chinensis (LC) on apparent digestibility, plasma parameters, ruminal fermentation, and microbial communities. Experimental treatments were four proportions of WCWH, 0, 33, 67, and 100%, as a substitute for LC (WCWH0, WCWH33, WCWH67, and WCWH100, respectively). The WCWH100 group showed a higher nutritional intake of crude protein (CP) and higher apparent digestibility of organic matter (OM), CP, and ether extract (EE) than the WCWH0 group (P P

Background Freemartinism generally occurs in female offspring of dizygotic twins in a mixed-sex pregnancy. Most bovine heterosexual twin females are freemartins. However, about 10% of bovine heterosexual twin females are fertile. Farmers mostly cull bovine fertile heterosexual twin females due to the lack of a practical diagnostic approach. Culling of such animals results in economic and genetic-material losses both for dairy and beef industry. Methods In this study, a comparative test, including qualitative detection of SRY gene by polymerase chain reaction (PCR), quantitative detection of relative content of SRY by real-time quantitative polymerase chain reaction (qPCR), and quantitative detection of H-Y antigen, was performed to establish the most accurate diagnosis for freemartin. Twelve Holstein heterosexual twin females were used in this study, while three normal Holstein bulls and three normal Holstein cows were used as a positive and negative control, respectively. Results Polymerase chain reaction results revealed that SRY gene were absent in three heterosexual twin females and only two of them were verified as fertile in later age. The qPCR results showed that relative content of SRY was more than 14.2% in freemartins and below 0.41% in fertile heterosexual twin females. The H-Y antigen test showed no significant numerical difference between freemartin and fertile heterosexual twin female. Discussion Our results show that relative content of SRY quantified by qPCR is a better detection method for diagnosis of freemartin in Holstein cattle as compare to qualitative detection of SRY gene by PCR or quantitative detection of H-Y antigen. To the authors’ knowledge, this is the first time we applied qPCR to diagnosing freemartin by quantifying SRY gene and got relative SRY content of each freemartin and fertile heterosexual twin female. We concluded that low-level of SRY would not influence fertility of bovine heterosexual twin female.