Embryonic development in repeat breeder and virgin heifers seven days after insemination

The aim of this study was to compare the development of embryos from repeat breeder heifers with that of embryos from virgin heifers at 7 days after standing heat. A total of 23 repeat breeder heifers (RBH) and 18 virgin heifers (VH) were utilized. The heifers were between 16 and 30 months of age and most of them were of the Swedish Red and White Breed. Two RBH were heterozygous for the $\text{1}\text{29}$ chromosome translocation, one RBH was a trisomy X and all the other heifers had normal karyotypes. All heifers were inseminated with frozen semen from the same bull and all inseminations were performed by the author. The fertility of the bull was above the average for the AI association to which it belonged. Embryos were collected by a non-surgical technique (89) or after slaughter (19). The morphology of the embryos was examined under a phase-contrast microscope and they were classified as being normal (N), morphologically deviating (MD) or degenerated (D). Thirteen embryos from RBH and 15 from VH were examined for total cell numbers after examination of their morphology.There was no significant difference in recovery rates of embryos between RBH (68%) and VH (76%) but independent of collection method the recovery rate of embryos from VH was numerically higher. The fertilization rate was high in both RBH (89%) and VH (97%). Seventyfour percent of the embryos collected from VH were normal ($\text{23}\text{31}$) while only 28% ($\text{11}\text{40}$) of the embryos collected from RBH had a normal morphology. The difference in number of normal embryos recovered from the two groups of heifers was highly significant (P < 0.005). Exclusion of the RBH heifers with deviating karyotype did not influence this difference. However, there was a tendency to a higher incidence of fertilization failure and morphologically deviating embryos in these heifers. The N embryos had significantly higher total cell numbers (P < 0.005) than the MD embryos but there was no significant difference in total cell numbers between N embryos from RBH or VH.The results of this study strongly indicate a higher incidence of abnormal embryos in RBH than in VH. It is likely that these deviations are followed by an increased incidence of early embryonic death.     

Reciprocal Embryo Transfer Between Repeat Breeder and Virgin Heifers — an Experimental Model

The aim of the study was to evaluate the importance of mother-concaptus relationships for the elevated embryonic loss in repeat breeder heifers. Embryos were collected by non-surgical technique, classified and transferred surgically or non-surgically to synchronized, inseminated recipients. The embryos were transferred to the uterinehorn contralateral to the corpus luteum. The embryos were transferred from repeat breeder heifers (RBH) to virgin heifers (VH) or from VH to RBH. After slaughter 4 weeks after transfer there was no difference in emhryonic survival between heifer categories following transfer or insemination. In some animals degenerated foetal membranes were found in the nonpregnant horn. The study indicates embryonic morphology rather than the category of donor or recipient as influencing the embryonic survival rate.     

Sequential endocrine changes and behaviour during oestrus and metoestrus in repeat breeder and virgin heifers

The aim of the experiment was to study the oestrous behaviour and the peripheral blood plasma profiles of luteinizing hormone (LH), progesterone and the prostaglandin metabolite, 15-keto-13,14-dihydro-PGF₂, during oestrus and metoestrus in repeat breeder (RBH) and virgin heifers (VH). Ten animals of each category were utilized. The RBH had a history of at least three inseminations without conception, and the VH were sexually mature animals not previously inseminated or mated. Oestrous symptoms were recorded and blood collected from the onset of prooestrus to 7 days after oestrus. The animals were inseminated during oestrus and their embryos were collected by a nonsurgical technique 7 days after insemination. The morphology of the embryos was evaluated.

The duration of oestrus was longer (P < 0.05) in the RBH (31.5 ± 3.6 h) than in the VH (23.8 ± 2.0 h). No differences in duration of prooestrus or in the interval from the end of oestrus to postoestrous bleeding were found between the heifer categories. The interval from the onset of oestrus to the preovulatory LH peak was longer (P < 0.05) in the RBH (12.2 ± 2.8 h) than in the VH (4.8 ± 1.5 h). There was a lower LH release in the RBH than in the VH, measured as the magnitude of the preovulatory LH peak (P < 0.05; 28.0 ± 4.0 vs. 40.7 ± 3.6 μg/l) or as the area under the curve of the LH peak (P < 0.01; 1141 ± 164 vs. 1765 ± 144 mm²). The progesterone levels were higher (P < 0.05) in the RBH than in the VH during the interval 5–48 h and lower (P < 0.05) during the interval 121–168 h after the LH peak. Peaks of the prostaglandin metabolite were seen during oestrus in both heifer groups. There were more prostaglandin metabolite peaks in the RBH than in the VH during the interval 13–24 h after the LH peak. Fewer normal embryos (P < 0.05) and more degenerated embryos (P < 0.01) were found in the RBH than in the VH group 7 days after insemination. No apparent relation was found between the morphology of the embryos and the hormonal changes.

The result of the study indicates a hormonal imbalance in the RBH. The hormonal asynchronism starts before or early in oestrus, which presumably leads to a sequence of improper hormonal changes responsible for the elevated embryonic loss in repeat breeder animals.     

Ovarian follicle apoptosis at the onset of standing estrus in virgin and repeat-breeder dairy heifers

There is evidence that repeat breeding in dairy cattle can be caused by both extrinsic, environmental factors and intrinsic, animal factors. In repeat-breeder heifers (RBH), disturbed endocrine patterns and estrous events result in a subsequent decreased fertility associated with delayed ovulation. Whether infertility is also due to the presence of an unsuitable follicular environment impairing normal fertilization, remains to be determined. At the onset of standing estrus, ovaries were obtained from 7 strictly defined RBH and 5 virgin heifers (VH) of the Swedish Red and White breed. Detection of apoptosis in the preovulatory and three subordinate follicle walls was done by using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) technique at light microscopy level. The follicles were histologically assessed for degree of atresia. The ultrastructure of the follicle wall and recovered oocytes was studied using transmission electron microscopy. The overall degree of apoptosis in membrana granulosa and theca interna of preovulatory and subordinate follicles did not differ between RBH and VH, but the numbers of TUNEL-positive cells differed significantly between preovulatory and subordinate follicles in both RBH and VH. There was a strong relationship between density of apoptotic cells and degree of atresia. No differences in follicle wall apoptosis nor morphology were detectable, suggesting that repeat breeder heifers enter standing estrus with the same morphological prerequisites as normal animals, considering follicular structure.     

Repeat breeding in dairy heifers: follicular dynamics and estrous cycle characteristics in relation to sexual hormone patterns

Repeat breeding occurs at an incidence of 10% in the Swedish dairy cow population.Evidence is available for a hormonal asynchrony around estrus in repeat-breeder heifers (RBH). This asynchrony seems to be the underlying cause for a series of dysfunctions such as prolonged standing estrus and delayed ovulation, leading to fertilization failure. For further determinations of repeat-breeder estrous cycle characteristics, seven strictly selected RBH and six virgin heifers (VH) were studied during 3-7 consecutive cycles, with particular attention paid to the estrous period. Follicular dynamics were studied by ultrasonography and related to estrous behavior and pattern of sexual hormones (progesterone, estradiol-17beta, and LH) in peripheral circulation. Mean group data were compared and a classification model was designed. The most prominent findings for RBH were prolonged duration of estrus, delayed LH peak, prolonged lifespan of the preovulatory follicle, and a late postovulatory rise in plasma progesterone. There was also a strong tendency for peri-ovulatory suprabasal progesterone levels in RBH. It is suspected that these deviations cause changes in the microenvironment of the preovulatory follicle, negatively affecting the final maturation of the oocyte. The heterogeneity of the RBH group underlines the multifactorial cause of the repeat-breeder syndrome. The VH formed a homogenous group with data varying within physiological limits. A classification model based on three characteristic variables managed to identify 81% of the VH and 79% of the RBH correctly. Results from this study propose that some heifers have general, consistent problems in synchronizing estrous events, displayed as varying symptoms in the course of consecutive estrous cycles. These subfertile animals could be classified as repeat-breeders.     

Effect of dietary protein on embryo recovery rate and quality in superovulated heifers

For almost 3 decades, superovulation and embryo transfer have been used in cattle breeding to increase the number of offspring from genetically superior female animals. Several factors including nutrition affect the number of transferable embryos recovered. We compared the effects of two different dietary protein levels easily achieved in practical conditions on embryo number and quality in superovulated heifers. Finnish Ayrshire heifers (n = 37) were allocated to isoenergic diets containing either 14% (D14) or 18% (D18) crude protein (CP). Estruses were synchronized, and the heifers were subsequently superovulated and inseminated using a standard FSH-protocol. Embryos were collected 7 days after inseminations (71-72 days after the beginning of the treatment period) by uterine flushing. The number of corpora lutea, and the number and quality of embryos were determined. Protein feeding did not affect superovulatory response, the number of embryos or the number of transferable embryos recovered. Proportionally more poor-quality embryos were found in group D14 than in group D18 (20.2% versus 13.2%, respectively, P = 0.053). It is concluded that a long-term moderate increase in the content of crude protein fed to energy-adequate heifers does not seem to affect superovulatory response and the number of embryos recovered, but it may be advantageous to the quality of embryos.     

Effect of ACTH-challenge on progesterone and cortisol levels in ovariectomised repeat breeder heifers

In order to investigate the potential influence of stress as a component of the repeat breeding syndrome, the adrenocortical capacity for steroid production was evaluated in ovariectomised dairy heifers. In repeat breeder heifers (RBH), marginally elevated plasma progesterone levels during oestrus, so-called suprabasal progesterone levels, have earlier been measured and are believed to impair fertility. The aim was to distinguish if this progesterone could be of extra-gonadal or in this case, adrenal origin. Baseline levels of plasma cortisol and progesterone were determined as well as the corresponding response after induced acute stress in the form of an adrenocorticotropin (ACTH)-challenge. Comparisons were made between strictly selected RBH, n=5 and virgin heifers (VH), n=5 of the Swedish Red and White breed. The heifers were used as their own pre-challenge controls in a 2-day trial. On the control day, saline was injected i.v. and on the treatment day, a synthetic analogue of ACTH (60 microg Synachten(R)). Via a jugular vein catheter, blood samples were collected every 30 min for 6 h each day of the experiment. Analyses for plasma progesterone and cortisol were made. RBH had a significantly higher (P<0.01) pretreatment baseline cortisol level (10.1+/-2.3 nmol l(-1)) than VH (2.6+/-0.2 nmol l(-1)). Moreover, the cortisol response after stimuli was stronger in RBH than VH, especially concerning total hormone production (P<0. 001), but there was also a tendency towards higher peak values (P=0. 06) and longer duration of significantly increased hormone concentrations (P=0.08). Progesterone concentrations, however, did not differ between the groups. Both baseline levels (P=0.25) and posttreatment production (P=0.45) were of the same magnitude in RBH and VH. In conclusion, the study could not confirm that suprabasal progesterone concentrations during oestrus in RBH derive from the adrenal glands. Still, apparent differences were found in adrenocortical activity when ovariectomised heifers, VH and RBH, were subjected to an ACTH-challenge. It is suggested that a sustained adrenal stimulation associated with environmental or social stress could be one factor in the repeat breeding syndrome.     

Embryonic development in superovulated dairy cattle exposed to elevated ambient temperatures between Days 1 to 7 post insemination

Holstein heifers (n = 29) were used to determine whether thermal stress during the first 7 d of embryonic development may increase the incidence of embryonic abnormalities in dairy cattle. Heifers were acclimated to environmental chambers at 20 degrees C for 9 d and superovulated with follicle stimulating hormone-pituitary (FSH-P; 40 mg total), beginning on Days 9 to 11 of the estrous cycle. Prostaglandin F(2)alpha (Lutalyse; 50 mg total) was administered on Day 3 of FSH-P. Heifers were inseminated artificially at estrus and then maintained at either thermal neutrality (20 degrees C) or under hyperthermic conditions (daily exposure up to 16 h at 30 degrees C and 8 h at 42 degrees C) for 7 d beginning at 30 h after the onset of estrus. On Day 7 post estrus, embryos were recovered nonsurgically and evaluated morphologically for stage of development and quality. The distribution of embryos classified as normal, abnormal, retarded or as unfertilized ova, differed (P<0.001) between heat stress and thermoneutral treatments. Only 20.7% of 82 embryos recovered from stressed heifers were normal compared with 51.5% of 68 embryos from thermoneutral animals. Stressed heifers had a higher incidence of abnormal and retarded embryos with degenerate nonviable blastomeres. Responses indicated that thermal stress from 30 h after the onset of estrus to Day 7 post estrus increases the incidence of abnormal and retarded embryos in superovulated heifers.     

Pregnancy Rates in Repeat-breeder Heifers Following Multiple Artificial Inseminations during Spontaneous Oestrus

Hormonal asynchronies during oestrus, related to the presence of suprabasal plasma-progesterone (P4) concentrations and a delayed ovulation, interfere with the fertility of repeat-breeder heifers (RBH). Since tubal dysfunction can occur in connection with hormonal asynchronies and constrained availability of fertile spermatozoa at the time of ovulation, the present study tested the hypothesis that frequent sperm deposition from onset of oestrus to ovulation may improve pregnancy rates in RBH. Five RBH and five virgin heifers (VH; controls) were repeatedly artificially inseminated (AI) at 6 h intervals from onset of oestrus to spontaneous ovulation. Hormone analyses revealed suprabasal P₄ concentrations and a delay in the occurrence of the luteinising hormone (LH) surge, but a normal cortisol profile in RBH. Compared with controls, RBH presented longer interval from onset of oestrus to ovulation, and therefore, received more AIs. Pregnancy rates in RBH reached control levels (60%; NS), indicating that the hypothesis might be correct. Pregnancy rates in VH were below the expected range, presumably attributed to a deleterious influence of the frequent handling. The study suggests that pregnancy rates can be improved in RBH by frequent AI in relation to spontaneous ovulation. However, this practice of repeated manipulations, while seeming not to show adverse effects, lacks practicality for routine use.     

Superovulatory response of dairy cattle (Bos taurus ) in a tropical environment

Dairy (Bos taurus) heifers and cows (n = 40) in a tropical environment were treated during mid-luteal phase using either SUPER-OV(R) or OVAGEN to induce superovulatory response after synchronization of the superovulatory estrus with a synthetic progestagen and cloprostenol (PG). Estrous cattle were inseminated twice using frozen-thawed semen, and embryos were recovered nonsurgically, on-farm, 7 d later. Between initiation of gonadotrophin treatment and recovery of embryos, 4 blood samples per animal were collected from 26 animals for determination of plasma progesterone (P4) concentration. Two (5%), 28 (70%) and 10 (22%) of the animals were observed in estrus 1.5, 2 and 2.5 to 3 d after PG, respectively. There was no difference (P = 0.7) in the number of palpable CL between animals treated with SUPER-OV (7.6 +/- 1.0; n = 18) and those treated with OVAGEN (7.9 +/- 1.1; n = 22). There was also no significant difference (P > 0.05) between Jersey vs Ayrshire breeds or heifers vs cows in the ovarian response as estimated by the number of palpable CL. However, a higher proportion of Ayrshire cattle and donors treated with OVAGEN yielded a higher total number and viable/transferable embryos than Jersey and SUPER-OV-treated cattle. There was a significant (P < 0.05) correlation between the number of CL and total number of embryos (r = 0.65); the number of transferable embryos was also significantly related to the total number of embryos per recovery (r = 0.85; P < 0.05). For 15 animals with normal P4 profiles, the mean (+/-SEM) plasma P4 concentration was 14.4 +/- 0.8, 0.5 +/- 0.2, 5.4 +/- 1.1 and 39.4 +/- 3.0 nmol L at initiation of gonadotrophin treatment, superovulatory estrus and Days 3 and 7, respectively. The mean (+/-SEM) interval between a PG injection given after embryo recovery and the induced estrus was 7.1 +/- 0.7 d (range 3 to 14 d) and the length of the superovulatory cycle was 24.1 +/- 3.2 d (range 12 to 35 d).     

The effect of co-treatment with recombinant bovine somatotrophin on plasma progesterone concentration and number of embryos collected from superovulated Holstein heifers

Mature Holstein heifers were induced to superovulate with twice-daily injections of porcine follicle-stimulating hormone (FSH), and were given either 20 mg i.m. of recombinant bovine somatotrophin (rBST) or saline with each FSH injection. The animals were artificially inseminated and the embryos were collected nonsurgically at Day 7. There was no significant difference in the mean (+/-S.D) total number of embryos collected from rBST-treated animals (8.3+/-5.3) when compared with that of the controls (7.2+/-6.6), or in the mean number of transferable embryos (5.3+/-4.0 vs 5.2+/-4.5). However, co-treatment with rBST tended to increase the ovulatory response, and it significantly increased plasma progesterone concentrations at Day 6 (P = 0.04). Based on these latter observations, we conclude that treatment with rBST enhanced the superovulatory response in heifers.     

Early pregnancy associated thrombocytopenia as an initial response to pregnancy in cattle

This study was conducted to determine if early pregnancy-associated thrombocytopenia exists in cattle as has been demonstrated in mice and in humans. Three experiments were designed to compare peripheral platelet counts in pregnant versus nonpregnant animals. In Experiment 1 heifers (n = 25) were artificially inseminated 12 h after the onset of estrus. Peripheral platelet counts in 19 pregnant versus 6 nonpregnant heifers did not reveal any significant differences between groups after insemination. In Experiment 2 embryos were collected nonsurgically from superovulated cows (n =18) on Days 6 to 7 after estrus. Platelet counts were monitored every 12 h after the first insemination until 60 h after the second insemination. Platelet counts and the number of embryos collected nonsurgically from these superovulated donors did not show any significant correlations (P>0.05). Ten recipient heifers synchronized to donor animals received either an unfertilized ovum or a good quality embryo via nonsurgical transfer into the uterus. There were no significant reductions in platelet counts after transfer. In Experiment 3 platelet counts were monitored daily in four pregnant and five nonpregnant recipient heifers between Day 0 and Day 30 after embryo transfer on Day 8 of the cycle. The platelet counts did not reveal any significant differences between the pregnant and nonpregnant groups throughout Days 0 to 30. These results indicate that early pregnancy-associated thrombocytopenia cannot be demonstrated in cattle. Peripheral platelet counts cannot be used as an indicator of early pregnancy in cattle.     

Plasma progesterone profiles and factors affecting embryo-fetal mortality following embryo transfer in dairy cattle

The relationship between plasma progesterone (P4) levels and embryo survival, and the value of P4 profiles for the selection of cattle embryo transfer recipients is still a matter of controversy. This study reports a comparison between lactating cows and heifers (n = 407) from a single dairy herd, after transfer of either fresh or frozen-thawed good quality embryos, of their ability to sustain embryo-fetal development to term. Plasma P4 concentrations on the day of estrus (Day 0 = D0), Day 4, Day 7 and on Day 21 were measured and related to embryo survival. Plasma P4 levels on Days 0, 4 and 7 were similar in recipients later found pregnant or open. Plasma P4 levels on Day 7 were significantly higher (P < 0.01) in heifers than in cows, but they were similar in pregnant and nonpregnant heifers and in pregnant and nonpregnant cows. Pregnancy rates for fresh and frozen-thawed embryos were higher in heifers than in cows, but the differences did not reach significance. However, the overall late embryonic mortality was significantly higher (P < 0.01) and the calving rate for frozen-thawed embryos was significantly lower (P < 0.05) in cows than in heifers. As expected, plasma P4 on Day 21 was significantly higher (P < 0.001) in pregnant than in nonpregnant recipients, but there was no difference between pregnant cows and pregnant heifers. Plasma P4 levels on Day 7 of recipients presumed pregnant on Day 21 and later found pregnant or nonpregnant were similar, but plasma P4 levels on Day 21 were significantly higher (P < 0.001) in pregnant than in nonpregnant recipients. The results of this study suggest that plasma P4 levels until the day of transfer, except for the rejection of recipients with abnormal luteal function, are of limited practical use for embryo transfer recipient selection. However, in lactating cows low plasma P4 values on Day 7 might negatively affect embryo survival, while in heifers this effect is not noticeable. Lactating cows are more prone to embryo loss than heifers, especially in the case of frozen-thawed embryos; this is associated with a lower competence of the corpus luteum at Day 7.     

The use of embryo collection techniques in Holstein heifers: A model to study early embryonic death

Twenty-three cyclic Holstein heifers were purchased for use as embryo donors to study the effect of intrauterine exposure to Haemophilus somnus on the number, quality, and viability of embryos produced. Few problems were encountered using standard superovulation and nonsurgical embryo collection techniques on virgin heifers. Based on three or more ovulations, as determined by palpation per rectum of the ovaries at the time of embryo recovery, 28 of 30 heifers responded to the superovulation regimen. Of 29 nonsurgical recoveries, 27 produced one or more embryos. One hundred and seventy-six embryos and ova were collected from heifers synchronized, superovulated, and flushed 7 to 8 d after insemination.     

The use of embryo transfer to produce pregnancies in repeat-breeding dairy cattle

Repeat breeding is an important factor affecting economic success in dairy management. The objective of this study was to investigate the effectiveness of transfer of frozen-thawed IVF embryos in establishing pregnancy in repeat-breeding Holstein cattle. Cumulus oocyte complexes were collected by aspiration of 2-5 mm follicles from ovaries obtained at two local abattoirs. After IVF, days 7 and 8 blastocysts were frozen either in 1.5M ethylene glycol with 0.1M sucrose, or in 1.4M glycerol with 0.1M sucrose. Holstein recipients (122 heifers and 410 cows) included those that had not conceived after 3-21 inseminations. Embryos frozen in ethylene glycol were transferred directly, and embryos frozen in glycerol were transferred after dilution of the cryoprotectant in sucrose into recipients 7 or 8 days after estrus (without-AI group), or following AI (with-AI group). Pregnancy rates were compared by the Chi-square test. Significantly higher pregnancy rates were achieved by embryo transfer following AI (with-AI group) than by embryo transfer alone (without-AI group) in both heifers (49.2 and 29.5%, respectively) and cows (41.5 and 20.4%, respectively; P<0.05). Pregnancy rates were not significantly different between heifers and cows. However, pregnancy rate decreased as the number of inseminations prior to embryo transfer increased in the with-AI group, but not in the without-AI group. Therefore, transfer of frozen-thawed IVF embryos during the same cycle in which AI was done improved pregnancy rates in repeat-breeding Holstein heifers and cows, and suggested that embryo transfer is an alternative in the treatment of repeat breeding.     

Gamete recovery and follicular transfer (graft) using transvaginal ultrasonography in cattle

Current in vitro culture systems may not be adequate to support maturation, fertilization and embryo development of calf oocytes. Thus, we initiated a study to investigate an alternative method of assessing oocyte competence in vivo, initially using oocytes from adults. Experiment 1 was done to determine if follicle puncture would alter subsequent follicle development, ovulation and CL formation. In control (no follicle puncture, n = 3) and treated (follicle puncture, n = 3) heifers, ultrasound-guided transvaginal follicle aspiration was used to ablate all follicles > or = 5 mm at random stages of the estrous cycle to induce synchronous follicular wave emergence among heifers; PGF2 alpha was given 4 d later. Three days after PGF2 alpha, the preovulatory follicle in treated heifers was punctured with a 25-g needle between the exposed and nonexposed portions of the follicular wall, and 200 microL of PBS were infused into the antrum. There was no significant difference between control and treated heifers for mean diameter of the dominant follicle prior to ovulation, the interval to ovulation following PGF2 alpha, or first detection and diameter of the CL. Experiment 2 was designed to assess multiple embryo production following interfollicular transfer of oocytes (i.e., transfer of multiple oocytes from donor follicles to a single recipient preovulatory follicle). Follicular wave emergence was synchronized among control (no follicle puncture, n = 5), oocyte recipient (n = 7) and oocyte donor (n = 5) heifers as in Experiment 1. In control and oocyte recipient heifers, a norgestomet ear implant was placed at the time of ablation and removed 4 d later, at the second PGF2 alpha treatment. In oocyte donor heifers, FSH was given the day after ablation, and, 4 d later, oocytes were collected by transvaginal follicle aspiration, pooled and placed in holding medium. Five or 6 oocytes were loaded into the 25-g needle of the follicle infusion apparatus with < or = 200 microL of transfer medium. Puncture of the preovulatory follicle of recipient heifers was done as in Experiment 1. Immediately thereafter, LH was given to control and oocyte recipient heifers, but only the recipients were inseminated. Ovarian function was assessed by transrectal ultrasonography and control and oocyte recipient heifers were sent to the abattoir 2 or 3 d after ovulation, where excised oviducts were flushed. The interval between LH administration and ovulation (33 to 36 h) was highly synchronous within and among control and oocyte recipient heifers. Four of 5 (80%) ova were collected from controls and 16 of a potential 43 (37%) ova/embryos were recovered from oocyte recipients; 8 embryos from 3 heifers. Thus, the gamete recovery and follicular transfer procedure (GRAFT) did not alter ovulation or subsequent CL formation, and resulted in the recovery of multiple ova/embryos in which a total of 19 oocytes yielded as many as 8 early embryos, a 42% embryo production rate.     

In vitro fertilization and culture of ova from heifers infected with bovine herpesvirus-1 (BHV-1)

Oocytes collected from heifers infected experimentally with bovine herpesvirus-1 (BHV-1, 10(8) TCID(50)/ml) and from dexamethasone-treated (stressed) BHV-1 seropositive animals were matured, fertilized and co-cultured in vitro for 7 d prior to being tested for the presence of the virus. Nineteen of the 21 infected donors yielded embryos and follicular fluids that were BHV-1 positive. Oviductal cells (17 21 ) and uterine fluids (14 21 ) were also positive. Titers for the positive samples ranged 10(1.6)-10(9.6) TCID(50)/ml. The cleavage rate and the proportion of blastocysts that developed from oocytes of BHV-1 infected animals were 26% (n=361) and 6% compared with 56% (n=112) and 26% for uninfected control donors (P<0.05). In contrast, embryos produced from dexamethasone-treated animals tested negative for BHV-1 and yielded 11% blastocysts as compared with 25% for the control group. The results indicate that transferable-stage embryos can be produced by IVF from infected BHV-1 animals and that such embryos are associated with the virus, and might have potential for disease transmission.     

Embryo production by ovum pick up in unstimulated calves before and after puberty

The possibility of producing embryos from oocytes repeatedly collected from unstimulated calves was tested, and results obtained before and after puberty were compared in the same animals. Ovum pick-up (OPU) coupled with in vitro embryo production was used on 2 sets of 7 and 9 calves, aged 7 to 10 m.o. at the start of the experiment. The oocytes were collected twice a week during a 2-m.o. period before puberty and a 1-m.o. period after puberty. Oocytes were fertilized and co-cultured with cumulus cells in modified synthetic oviduct fluid (SOF) up to Day 7 post insemination. Some Day 7 blastocysts were vitrified and transferred to recipient heifers. An average of 3.8 to 6.8 follicles was punctured per OPU session; 1.9 to 3.1 oocytes were collected, of which more than 60% were of good quality. The number of punctured follicles and collected oocytes varied between donors. Blastocyst rates of 19 to 27% were obtained for the 2 sets. Three normal calves were born from the transfer of 20 vitrified embryos. While no significant difference was observed for the first set of calves, a significant decrease in the number of punctured follicles was observed after puberty in the second set. A direct correlation was also obtained between the number of follicles punctured before and after puberty in the same animal. In conclusion, oocytes can be collected by repeated OPU in calves 7 to 10 m.o. old without affecting their growth or the onset of puberty. An average of 5 to 11 (range 0 to 16) blastocysts per donor was produced over 2 month. However, important variations were found between donors. The correlation observed for the number of follicles punctured before and after puberty suggests that this parameter is determined before puberty.     

Embryo yield and plasma progesterone profiles in superovulated dairy cows and heifers.

This study was conducted to compare the superovulatory (SOV) response of dairy cows (n=172) and heifers (n=172), with two SOV treatments started at the mid-luteal-phase of the estrus cycle. Donors were randomly treated either with equine chorionic gonadotrophin (eCG) plus neutra-eCG serum (eCG+N group, n=167) or follicle stimulating gonadotrophin (FSH-P group, n=177).No significant differences were observed among groups in the percentage of superovulatory responsive donors (SR donors; corpora lutea (CL) >/=2), the mean number of total ova, fertilized ova and viable embryos recovered. Cows yielded significantly less total ova and less fertilized ova (P<0.05) and tended to yield less viable embryos (P<0.06) than heifers.Plasma progesterone (P4) concentrations (n=135 donors) on the day of PGF(2alpha) (PGF) injection and on the day of SOV estrus were significantly higher (P<0.01) in eCG+N than in FSH-P donors and, the increase between those 2 days was also significantly higher (P<0.05) in group eCG+N than in group FSH-P, suggesting a higher luteotrophic effect of eCG than FSH-P. SR donors had P4 levels significantly higher (P<0.001) than non-SR donors only on day 5 after the SOV estrus and on the day of embryo recovery. Plasma P4 concentrations at 5 days after the SOV estrus and at embryo recovery correlated significantly (r=0.76, P<0.001).Heifers had significantly higher P4 levels than cows at gonadotrophin injection (P<0.01), PGF injection (P<0.001), 5 days (P<0.01) and 7 days (P<0.001) after the SOV estrus. At day 7 after the SOV estrus, P4 concentrations per ova recovered were significantly higher in heifers than in cows (P<0.01). The increase of plasma P4 per ova recovered, between days 5 and 7 after the SOV estrus, was significantly (P<0.01) higher in heifers than in cows. Also, the increase of plasma P4 between injections of gonadotrophin and PGF was significantly higher (P<0.05) in heifers than in cows.These results suggest that heifers have higher plasma P4 concentrations at diestrus (either before or after the SOV treatment) and this is associated with a higher embryo yield and quality, as compared to lactating cows. These higher plasma P4 concentrations reflect not only differences in ovulation rate as well as the competence of the corpus luteum, which is potentialized by gonadotrophin stimulation.     

Relationship between plasma progesterone concentrations and pregnancy rates in cattle receiving either fresh or previously frozen embryos

Blood samples were collected for the measurement of progesterone concentrations from 320 Holstein-Friesian heifers on Days 7 and 21 post-estrus. All animals were the recipients of either a fresh or previously frozen embryo on Day 7 and were palpated for pregnancy on Day 60 post-estrus. At the time of transer, progesterone levels were highly variable and were not strongly related to subsequent pregnancy status. There was a tendency for lower pregnancy rates in heifers receiving fresh embryos if progesterone levels were less than 1 ng/ml (33 vs 64% overall), and for previously frozen embryos when progesterone concentrations were less than 3 ng/ml (34 vs 44% overall). Progesterone concentrations were not related to subjective evaluation of corpus luteum quality by palpation per rectum. No heifers which maintained pregnancy had progesterone levels less than 1 ng/ml on Day 21. Only 41% of 247 heifers receiving either fresh or previously frozen embryos that were not pregnant on Day 60 had progesterone concentrations less than 1 ng/ml on Day 21. These data suggest that many recipients that do not maintain a pregnancy will experience an extended estrous cycle after transfer.