Induction of twinning in crossbred heifers by ipsilateral frozen embryo transfer

Induction of twinning by means of nonsurgical ipsilateral transfer of two frozen embryos or of demi-embryos was attempted in 297 virgin crossbred dairy heifers that were kept at the following locations: in stables at our breeding station (Group 1), in a mountain field (Group 2) and at a private farm (Group 3). Of these, 187 heifers (63%) were diagnosed pregnant at 25 d after embryo transfer and 134 heifers (45%) were diagnosed pregnant at 60 d after transfer by ultrasonic echography or by palpation per rectum. At calving, 118 heifers produced 46 sets of twins and 72 single calves, for a total of 164 calves sixteen pregnant heifers aborted fetuses between 2 and 7 mo. The pregnancy rate of Group 1 (39%) was lower than that of Group 2 (48%) or Group 3 (50%). Abortion and mortality rates for Group 3 (8% and 8%, respectively) were significantly lower (P<0.05) than for Group 1 (16% and 12%) or Group 2 (13% and 18%). Twin calves had lower birth weights (P<0.05) than singles, but at 270 to 330 d of age, there was no significant difference between the weights of single and twin calves. Dystocia or difficult delivery was not observed heifers producing twins. Retention of the placenta was observed in twin calving heifers, with placenta retention levels the highest in Group 2 (33%) than followed by Group 1 (14%) and Group 3 (10%). When gestation periods were compared between single and twin calves in Groups 1 and 2, the gestation period of single calves was 5.0 to 7.0 d shorter than that of twin calves. No significant difference was observed between the gestation periods of single and twin births in Group 3.     

Minimal progesterone concentration required for embryo survival after embryo transfer in lactating Holstein cows

Objectives were to determine progesterone concentration (P4) from days 4 to 28 relative to presumptive estrus necessary for maintenance of pregnancy in lactating Holstein cows. Cows were assigned to the low P4 (LowP4, n = 28) or control (n = 153) treatments. All cows were presynchronized with two injections of PGF_2α (14 d apart) and an ovulation synchronization protocol (11 d later; GnRH on day −10, PGF_2α on day −3; and GnRH on day 0 = presumptive estrus). Cows in the Low P4 treatment received 2 injections of prostaglandin F_2α on days 4 and 5 (day 0 = presumptive estrus) and a new CIDR insert on day 5 that was replaced every 7 d until day 28. Blood was sampled on days −9, −2, 0, 4, 7,14, 21, and 28. Ovaries were examined with ultrasound on days −9, −3, and 7 and cows bearing a corpus luteum ≥20 mm on day 7 received an embryo. On days 0, 4 and 7 P4 did not differ (P ≥ 0.27) but control cows had greater (P < 0.01) P4 on days 14, 21, and 28. Progesterone concentration fold change from day 0 to 7 was not (P = 0.14) affected by treatment, but P4 concentration fold change from day 7 to 14 was (P < 0.01) greater for control cows compared with LowP4 cows. No LowP4 cows became pregnant after embryo transfer, whereas 35.7, 25.5, and 21.4% of control cows were pregnant on day 28, 42, and 63, respectively. Progesterone concentration fold changes from day 0 to 7 (P = 0.03) and from day 7 to 14 (P = 0.05) were associated with pregnancy outcomes on day 63. Among cows that were pregnant on day 63, the minimum P4 concentration fold changes from day 0 to 7 and from day 7 to 14 were 2.71 and 1.48, respectively. Interestingly, cows with P4 concentration <5 ng/mL on day 14 were (P = 0.01) and tended to be (P = 0.07) more likely to lose pregnancy from day 28 to 42 and from day 28 and 63, respectively. Faster rise in P4 concentration during the metestrus and early diestrus are associated with pregnancy establishment following embryo transfer, which suggests that early rise in P4 concentration has an indirect effect on embryo development through modulation of uterine environment and secretion of histotroph. Furthermore, the positive effects of early rise in P4 concentration appear to go beyond the phase of maternal recognition of pregnancy through adhesion and placentation stages.     

Artificial induction of twinning in cattle by means of supplemental embryo transfer

The induction of twins by surgical and nonsurgical transfer was performed 6 - 8 days after Artificial Insemination (A.I.) in a total of 142 cows. Of the 47 heifers, which received surgically transferred embryos, 32 (68%) became pregnant. Of the 95 cows, which received nonsurgically transferred embryos, 58 (61,6%) became pregnant. A total of 90 (63.5%) of the recipient animals became pregnant. Up to date 64 recipients have calved; twins have resulted in 40 and 48,2% resp, from surgical and nonsurgical inductions. Out of a total of 64 calvings 29 (45,3%) were twins, and of these 38% were males, 27% were females, and 34% were mixed births. Female - calves resulted in freemartinism. There was no increase in the incidence of either retained placenta or dystocia. The induction of twinning by means of embryo transfer can be regarded as a useful method for the increase in the reproductive performance cattle.     

Superovulation and embryo transfer in Holstein cattle using sexed sperm

The use of sexed bull sperm in multiple ovulation and embryo transfer (MOET) programs for Holsteins was evaluated for (1) heifers housed at a commercial embryo transfer (ET) facility (Experiments 1 and 2), and (2) heifers and cows on dairy farms (Experiment 3). In Experiment 1, superstimulated heifers were inseminated with 5 × 10⁶ sexed (X-sorted; n = 5) or unsexed (n = 5) frozen-thawed sperm from one bull at 12 and 24 h after estrus detection. No difference was observed in the rates of transferable embryos (53.4% vs 68.1%), degenerate embryos (24.8% vs 26.6%) and unfertilized ova (21.8% vs 5.3%) between sexed and unsexed sperm, respectively, except for the percent of female transferable embryos diagnosed by embryo sexing (100% vs 49.3%, P < 0.0001). In Experiment 2, donors were inseminated twice with 5 × 10⁶ sexed unfrozen sperm (n = 10) or sexed frozen-thawed sperm (n = 9). Embryo production rates for both treatments were similar to that observed on a commercial ET facility using unsexed sperm. Pregnancy rates for frozen-thawed embryos were similar for sexed and unsexed sperm (70.4% vs 72.4%, respectively). In Experiment 3, 99 flushes were conducted using sexed frozen-thawed sperm from nine bulls but an overall statistical analysis was not completed because the use of bulls was not balanced. However, for one bull with balanced usage, the rate of transferable embryos was higher in heifers than in cows (P < 0.05) inseminated twice with 5 × 10⁶ sperm/dose (10 × 10⁶ total). We concluded that the use of sexed frozen-thawed sperm (≥90% X-sperm biased and 10 × 10⁶ total sperm) may be economically viable for commercial MOET programs in Holstein heifers.     

Calf survival from embryo transfer-induced twinning in dairy-beef cows and the effects of synchronised calving

In each of 4 years, 94-116 mature cows had two 6-7-day-old embryos, produced by the in vitro fertilisation of oocytes, inserted non-surgically into one uterine horn of each cow. Starting 5 days before the expected date of calving, the cows were continuously observed and assistance at calving was provided when required. In year 1, perinatal calf survival was similar in twin-calving (TC) and single-calving (SC) cows (98.1 versus 100% for calves born to TC and SC, respectively). There was a higher incidence of assistance at birth for TC (52%) than for SC (21%). In years 2 and 3, the calving of 30 SC and 33 TC was synchronised using an injection of Opticortinol (OP) 6-9 days before the injection of Estrumate and Dexol-5 (E+D). A further 34 SC calved naturally. Synchronised calving reduced the spread of calving from 16-25 to 8-9 days without reducing perinatal calf survival and had no significant effect on the incidence of assistance at birth in SC. The TC in years 2 and 3 had a high incidence of retained placenta at 48 h (70%) and a high incidence of assistance at birth (85%). In year 4, calving was synchronised in 16 SC and 21 TC with E+D and no pre-treatment with OP, while 15 SC were treated with both OP and E+D. There were no effects of the hormone treatment on perinatal calf survival and only small effects on the incidence of assisted births for SC. The incidence of retained placenta at 48 h was lower for SC pre-treated with OP (40%) than for SC (88%) and TC (76%) not pre-treated with OP. Continuous supervision over calving produced perinatal calf survival rates for TC that were similar to SC, despite the higher incidence of assistance of TC at parturition. Hormonal synchronisation of calving can halve the time required for continuous supervision of calving, but the hormone treatments exacerbate the already high incidence of retained placenta in TC.     

Effects of rumen-protected methionine and choline supplementation on the preimplantation embryo in Holstein cows

Our objective was to determine the effects of supplementing methionine and choline during the prepartum and postpartum periods on preimplantation embryos of Holstein cows. Multiparous cows were assigned in a randomized complete-block design into four treatments from 21 days before calving to 30 days in milk (DIM). Treatments (TRT) were MET (n = 9, fed the basal diet + rumen-protected methionine at a rate of 0.08% [w:w] of the dry matter [DM], Smartamine M), CHO (n = 8, fed the basal diet + choline 60 g/d, Reashure), MIX (n = 11, fed the basal diet + Smartamine M and 60 g/d Reashure), and CON (n = 8, no supplementation, fed the close-up and fresh cow diets). Cows were randomly reassigned to two new groups (GRP) to receive the following diets from 31 to 72 DIM; control (CNT, n = 16, fed a basal diet) and SMT (n = 20, fed the basal diet + 0.08% [w:w] of the dry matter intake as methionine). An progesterone intravaginal insert (CIDR) device was inserted in all cows after follicular aspiration (60 DIM) and superovulation began at Day 61.5 using FSH in eight decreasing doses at 12-hour intervals over a 4-day period. On Days 63 and 64, all cows received two injections of PGF2α, and CIDR was removed on Day 65. Twenty-four hours after CIDR removal, ovulation was induced with GnRH. Cows received artificial insemination at 12 hours and 24 hours after GnRH. Embryos were flushed 6.5 days after artificial insemination. Global methylation of the embryos was assessed by immunofluorescent labeling of 5-methylcytosine, whereas lipid content was assessed by staining with Nile red. Nuclear staining was used to count the total number of cells per embryo. There was no difference between TRT, GRP, or their interaction (P > 0.05) for embryo recovery, embryos recovered, embryo quality, embryo stage, or cells per embryo. Methylation of the DNA had a TRT by GRP interaction (P = 0.01). Embryos from cows in CON-CNT had greater (P = 0.04) methylation (0.87 ± 0.09 arbitrary units [AU]) than embryos from cows in MET-CNT (0.44 ± 0.07 AU). The cytoplasmic lipid content was not affected (P > 0.05) by TRT or their interaction, but lipid content was greater (P = 0.04) for SMT (7.02 ± 1.03 AU) than that in CNT (3.61 ± 1.20 AU). In conclusion, cows in MET-CNT had embryos with lower methylation, and SMT cows had a higher lipid content than CNT. Methionine supplementation seems to impact the preimplantation embryo in a way that enhances its capacity for survival because there is strong evidence that endogenous lipid reserves serve as an energy substrate.     

Seasonal effects on embryo transfer results in Brahman cows

To determine factors affecting embryo donor reproductive performance in Bos indicus cows, 1,841 embryo collection records of 813 Brahman donor cows were analyzed using least-squares methods for unequal subclass numbers. The basic model included the main effects of either season or month, technician, and year of collection. Season and year did not affect (P>.10) the variance of number of ova collected. The most skillful technician collected 2.8 more ova per flush compared with the least skillful. Number of transferable embryos recovered per donor cow was affected by season (P<.06). Maximal recovery of transferable embryos occurred during the fall season (4.2) with a lower number (2.9) recovered during the winter season. Pregnancy rate in the recipients followed a seasonal pattern with lower (31.8%) pregnancy rates during the winter and higher (41.0%) ones in the fall. There was a trend toward higher pregnancy rates during February through October and lower rates December and January. Number of blastocysts recovered per donor was affected by season (P<.08), and year (P<.01). Number of blastocysts recovered per donor was lower (2.0) during the winter season than during the fall season (3.1). These data establish the concept of seasonal effects on embryo donor reproductive performance in Brahman cows.     

Production and health assessment of second-generation cloned Holstein cows derived by somatic cell nuclear transfer

In this study we evaluated cloning efficiency of second-generation (G2) cloned Holstein cows derived from ear fibroblasts of a first-generation (G1) cloned cow, and assessed their health status in terms of physical, growth and reproductive parameters. Compared with G1 cloning, G2 cloning showed a slight decrease on blastocyst rate of reconstructed embryos (30.2±5.8% vs. 28.5±7.2%, p>0.05), while the quality of its blastocysts reduced significantly (Grade 1 and Grade 2, 21.1±4.1% vs. 17.1±5.7%, p<0.05). After embryo transfer (ET), both pregnancy rate to term and calving rate of G2 cloning were approximately half of G1 cloning (5.8% vs. 10.7%; 3.9% vs. 8.6%, p>0.05). Six G2 cloned cows were delivered, and three of them survived. G2 cloned calves displayed symptoms of being overweight at birth and tachycardia in the first week after birth. During the first 12 months, the growth of G2 cloned calves was similar to control calves derived from artificial insemination (AI). Furthermore, the interindividual variation of growth within the G2 clonal family was smaller except at birth and at two months of age. Interestingly, although G2 cloned cows reached puberty 45 days later in comparison with control cows derived from AI, they were all pregnant by AI, and gave birth to healthy calves. This suggests that their reproductive performance was not affected by late puberty. In summary, our results showed that although cloning efficiency of G2 was lower than that of G1, the surviving G2 clones appeared physically healthy and were fertile.     

Bio-economic model to evaluate twinning rate using sexed embryo transfer in dairy herds

A stochastic bio-economic model has been used to determine the effects of new reproductive technologies over a 15-year period. A strategy of using conventional artificial insemination (AI) or embryo transfer (ET) using two sex-controlled embryos at different conception rates (CRs) and herd sizes resulted in a 24 state model. The genetic means of AI population increased over the years, and the genetic means of milk production for all of the embryo strategies were greater than those of AI. In addition, the genetic means of milk yield using different embryo-based scenarios in the expanding herds were greater than those for the fixed herds. The net profit of using sexed ET in the expanding herds was greater (P < 0.05) than that of fixed size herds. In general, there was a roughly consistent trend in net profit per cow for sexed ET strategies in the expanding herds over the years, but there was an increasing trend in net profit per cow for sexed ET strategies in the fixed herds over the years. Medium to high CRs for ET and the use of sex-controlled embryo systems, especially for induction of twin births to produce dairy replacements, will be critical elements of a system that produces significant numbers of female calves. The greater number of female calves produced in the sex-controlled scenarios allows the farmer to select animals with the best genetic potential as dairy replacement heifers; therefore, the rate of genetic gain increased in the dairy herd. Results of sensitivity analyses showed that a significant decrease in the production costs and increase in the ET performance are essential for embryo-based technologies to be profitable.     

Induction of monozygotic twinning by ascorbic acid in tobacco

Embryo development in plants initiates following the transverse division of a zygote into an apical, proembryo cell and a basal cell that gives rise to the suspensor. Although mutants affected in embryo development through changes in cell division have been described, little is known about the control of the first zygotic division that gives rise to the proembryo. Ascorbic acid (Asc) promotes cell division by inducing G(1) to S progression but its role in embryo development has not been examined. In this study, we show that the level of dehydroascorbate reductase (DHAR) expression, which recycles Asc and regulates Asc pool size, affects the rate of monozygotic twinning and polycotyly. DHAR-induced twinning resulted from altered cell polarity and longitudinal instead of transverse cell division that generated embryos of equal size. Direct injection of Asc into ovaries phenocopied DHAR-induced twinning. Twinning induced by Asc was developmentally limited to the first two days after pollination whereas polycotyly was induced when the level of Asc was elevated just prior to cotyledon initiation. This work describes the first example of gene-directed monozygotic twinning and shows that Asc regulates cell polarity during embryo development.     

Ovarian responses and embryo survival in recipient lactating Holstein cows treated with equine chorionic gonadotropin

The objectives of Experiment 1 were to determine a dose of eCG that would increase total luteal volume and plasma progesterone (P4) concentration on estrous cycle Day 7 in cows. The objectives of Experiment 2 were to determine the effects of treating embryo recipient lactating Holstein cows with eCG on pregnancy per embryo transfer (P/ET). In Experiment 1, lactating dairy cows at 63 ± 3 d postpartum (DIM) received no treatment (control, n = 10), or 600 (eCG6, n = 19), or 800 (eCG8, n = 19) IU of eCG 2 d after the start of the ovulation-synchronization protocol, Day -8 (Day -10 GnRH, Day -3 PGF_2α, Day 0 GnRH). Blood was sampled on Days -10, -8, -3, 0, 7, and 14 for P4 concentration. Ovaries were examined by ultrasound on Days -10, -3, 0, and 7. In Experiment 2, lactating dairy cows were paired according to parity and previous insemination (0 or > 1 insemination) and assigned to receive 800 IU of eCG (eCG8, n = 152) 2 d after the start of the ovulation-synchronization protocol (Day -10 GnRH, Day -3 PGF_2α, Day 0 GnRH) or to receive no treatment (control, n = 162). Blood was sampled on Days -10, -3, 0, 7, and 14 for determination of P4 concentration. Ovaries were examined by ultrasound on Days -10, -3, and 7, and cows with a CL > 20 mm in diameter on Day 7 received an embryo. In Experiment 1, P4 concentration on Day 7 was higher (P < 0.05) for eCG8 cows (2.3 ± 0.3 ng/mL) compared with control (1.2 ± 0.3 ng/mL) and eCG6 (1.1 ± 0.3 ng/mL) cows. In Experiment 2, eCG8 primiparous cows had more (P < 0.01) follicles > 10 mm on Day -3 compared with control primiparous cows (2.5 ± 0.9 vs 1.7 ± 0.5 mm), but multiparous control and eCG8 cows did not differ. A larger (P = 0.03) percentage of control cows received an embryo (87.5 vs 79.1%) compared with eCG8 cows. Among cows that received an embryo, total luteal volume on Day 7 was affected (P = 0.05) by treatment (eCG8 = 8.3 ± 0.4 cm³, control = 6.2 ± 0.4 cm³), but P4 concentration on Day 7 did not differ significantly between treatments. The percentage of cows pregnant 53 d after ET (overall, 24.2%) was not significantly different between control and eCG8 cows. In the current study, no differences in P/ET were observed between control and eCG8 cows and treatment with eCG increased the percentage of cows with asynchronous estrous cycle.     

Factors affecting frozen and fresh embryo transfer pregnancy rates in cattle.

The effects of a large number of factors on the pregnancy rates of fresh and frozen cattle embryos were examined over a period of years at several different locations. For fresh embryos, overall pregnancy rates were 68.3% (n=9023) and 77.1% (n=2650) at different locations and time periods. Frozen-thawed embryo pregnancy rates were 56.1% (n=3616) in The Netherlands and 58.4% (n=5297) and 68.7% (n=774) for two studies in the United States. Pregnancy rates of surgical versus nonsurgical transfers were very similar. There were no differences in the pregnancy rates of beef versus dairy embryos, but the pregnancy rate was higher in dairy and beef heifers and beef cows than in dairy cows. Although on-farm pregnancy rates in California were higher than in the northeast United States, there was no influence of season on pregnancy rate. Estrous asynchrony between plus and minus 24 h did not affect pregnancy rate for frozen-thawed or fresh embryos. Neither breed nor parity of recipients affected the influence of asynchrony on pregnancy rates. Embryo grade was a significant factor in pregnancy rate for both fresh and frozen-thawed embryos, but neither embryo stage nor age was a significant factor. Pregnancy rate was not affected by holding embryos after flushing for up to 3 h prior to freezing.     

Comparison of artificial insemination versus embryo transfer in lactating dairy cows

Conception rates (CR) are low in dairy cows and previous research suggests that this could be due to impaired early embryonic development. Therefore, we hypothesized that CR could be improved by embryo transfer (ET) compared with AI. During 365 days, 550 potential breedings were used from 243 lactating Holstein cows (average milk production, 35 kg/day). Cows had their ovulation synchronized (GnRH-7d-PGF(2alpha)-3d-GnRH) and they were randomly assigned for AI immediately after the second GnRH injection (Day 0) or for transfer of one embryo 7 days later. Circulating progesterone concentrations and follicular and luteal size were determined on Days 0 and 7. Pregnancy diagnosis was performed on Days 25 or 32 and pregnant cows were reevaluated on Days 60-66. Single-ovulating cows with synchronized ovarian status had similar CR on Days 25-32 with ET (n = 176; 40.3%) and AI (n = 160; 35.6%). Pregnancy loss between Days 25-32 and 60-66 also did not differ (P = 0.38) between ET (26.2%) and AI (18.6%). When single (n = 334) and multiple (n = 57) ovulators were compared, independent of treatment, multiple ovulators had greater (P < 0.001) circulating progesterone concentrations on Day 7 (2.7 ng/ml versus 1.9 ng/ml) and there was a tendency (P = 0.10) for a greater CR in multiple ovulators (50.9% versus 38.1%). However, there was no difference in CR between AI and ET cows with multiple ovulations (50.0% versus 51.7%). In single-ovulating cows, CR tended to be lower for AI than ET in cows ovulating smaller follicles (diameter < or = 15 mm; 23.7% versus 42.3%; P = 0.06) but not average-diameter follicles (16-19 mm; 41.2% versus 37.3%; P = 0.81) or larger (> or =20 mm; 34.3 versus 51.0%; P = 0.36) follicles. Thus, although ET did not improve overall CR in lactating cows, follicle diameter and number of ovulating follicles may determine success with these procedures.     

Observed frequency of monozygotic twinning in Holstein dairy cattle

Bonnier's equation is used to mathematically estimate the frequency of monozygotic (MZ) twinning in epidemiologic studies of twinning in dairy cattle; however, no empirical determination of MZ twinning has been reported in the literature. Our objectives were to empirically determine the frequency of MZ twinning in lactating Holstein cows and to compare this result with published estimates predicted using Bonnier's equation. Ear biopsies were collected from 107 sets of Holstein twins from six Wisconsin dairies resulting in 40 opposite-sex twins, 29 same-sex male twins, and 38 same-sex female twins. To empirically determine the frequency of MZ twinning, DNA extracted from ear biopsies collected from the 67 same-sex twins was PCR amplified using primers for a minimum of 5 polymorphic microsatellite DNA markers. Opposite-sex twins were classified as dizygotic (DZ) as well as same-sex twins differing in at least one microsatellite DNA marker. Same-sex twins were classified as MZ when all genotypes for a minimum of five markers were identical. Of the 67 same-sex twins, 62 were classified as DZ and 5 MZ resulting in a MZ twinning frequency of 7.5% of same-sex twins and 4.7% of all twins. The estimated frequency of MZ twinning in this population of twin calves using Bonnier's equation was 39.5% of same-sex twins and 24.7% of all twins. We concluded that MZ twinning occurred infrequently in Holstein cattle and perhaps less frequently than that reported in studies using Bonnier's equation to estimate MZ twinning.     

Effects of sex control and twinning on economic optimization of culling cows in Japanese Black cow-calf production systems

The effects of sex control and twinning techniques on determination of optimal culling parity of cows in beef cow-calf production systems were deterministically analyzed using a herd model simulation. The model simulated the annualized net revenue as an economic indicator during the whole life cycle of a cow. Biological factors (survivability, growth, reproduction, and feed requirements) and economic factors (returns from sales of live calves and cows' carcasses and production costs) were included in the model. Some biological and economic parameters relating to these factors were altered from a base condition in order to adapt the production systems with sex control and twinning techniques. Based on the model, early culling was optimal for all production systems when biological efficiency was used as an indicator of production; however, later culling was optimal for single production, but slightly earlier culling was optimal for twin production, when annualized net revenue was evaluated. The introduction of sex control did not greatly affect the determination of the optimal culling parity of cows. When production included the sex control, female sexing increased biological efficiency, whereas male sexing increased annualized net revenue. In the present beef cow-calf production circumstances in Japan, introduction of sex control did not have economically appreciable effects, but twinning was economically beneficial. For production involving sex control, improvement in the conception rate per mating and/or reduction of technical cost were required for this technology to be profitable.