Short intervals between ultrasonographically guided follicle aspiration improve oocyte quality but do not prevent establishment of dominant follicles in the Gir breed (Bos indicus) of cattle

The aim of the present study was to evaluate oocyte recovery and embryo yield using two different ovarian follicular aspiration schedules in donor cows of the Gir breed. Pluriparous, non-lactating Gir cows (n = 14) were randomly allocated to one of two groups, one of which had aspirations of ovarian follicular contents conducted once a week (TVFA-1x) and the other twice weekly (TVFA-2x), for nine consecutive weeks. Before follicle aspiration, follicular population was recorded in three classes according to size (> 6 mm, 6-9 mm and > 9 mm). The cumulus-oocyte complexes (COCs) recovered were identified, morphologically classified and in vitro matured, fertilized with Gir sperm and cultured in CR2 medium for 7 days. There was no difference (P > 0.05) in the size of the largest follicle, number of follicles identified or follicular content aspirations between TVFA-1x and TVFA-2x groups. Large follicles (> 9 mm) were observed for all the aspiration intervals considered (3, 4 or 7 days). More oocytes were recovered per session in TVFA-1x as compared with TVFA-2x (8.9 +/- 0.8 versus 7.0 +/- 0.7, P < 0.01), resulting in a greater recovery rate in this group (74.3% versus 58.7%, P < 0.01). More COCs of Grade I were recovered from TVFA-2x (22.6% versus 13.3%, P < 0.01). There was no difference in cleavage rate between groups, but the percentage of embryos that reached the blastocyst stage was greater in TVFA-2x as compared with the TVFA-1x (31.8% versus 21.6%, P < 0.01). The greater in vitro performance qualities of TVFA-2x oocytes compensates for the greater oocyte recovery rate in TVFA-1x, demonstrating a greater embryo production potential. Despite showing uncommon follicular dynamics characteristics when subjected to follicular aspiration, Gir cows can be successfully used as oocyte donors for in vitro embryo production.     

Ultrasound image characteristics of ovarian follicles in relation to oocyte competence and follicular status in cattle

Assessment of the quality of the female gamete has become paramount for in vitro procedures. There is a need to identify reliable indicators of oocyte competence and develop a simple, non-invasive method to assess competence. The aim of this study was to investigate the relationships among ultrasonographic attributes of a follicle, its stage of development and the competence of the oocyte that it contains. We tested the hypotheses that follicular echotexture characteristics are related to: (1) the phase of development of the follicle, (2) the presence of the corpus luteum (CL) and/or the dominant follicle in the ovary, and (3) developmental competence of cumulus oocyte complexes (COC) from the same ovary. Crossbred beef cows (n=143), age 4-14 years, were given a luteolytic dose of dinoprost to cause ovulation. Ultrasound-guided ablation of all follicles > or = 4mm was done 8 days later to induce new follicular wave emergence during a luteal phase. Ultrasonographic images of dominant follicles and the three largest subordinate follicles (n=402 follicles; 84 cows) were acquired on Days 2, 3, 5 or 7 of the follicular wave (Day 0: wave emergence), i.e. growing, early-static, late static, and regressing phases of subordinate follicle development, respectively. From a subset of these animals (n=33), ovaries were collected within 30 min of slaughter and COC from subordinate follicles > or = 3mm underwent in vitro maturation, fertilization and culture to the blastocyst stage.Image analysis revealed differences in echotexture between dominant and subordinate follicles among Days 2-7 of the follicular wave. Images of dominant and subordinate follicles at Day 7 of the wave displayed consistently lower grey-scale values (P<0.05) in the peripheral antrum, follicular wall and perifollicular stroma than all other days. Follicle images displayed a consistent pattern of variation in echotexture among follicular phases. Data did not support the hypothesis of a local effect of the CL or dominant follicle on follicular echotexture. Echotexture values of the perifollicular stroma were lower in ovaries that did not produce embryos compared to ovaries that produced embryos. Our results showed that the changes in follicular image attributes are consistent with changes in follicular status. The sensitivity of the technique is not yet sufficient for use in a diagnostic setting, but results provide rationale for further development of image analysis as a tool for evaluating oocyte competence in situ.     

The effect of feeding propylene glycol to dairy cows during the early postpartum period on follicular dynamics and on metabolic parameters related to fertility

Postpartum dairy cows (n=35) were used to determine the effects of feeding propylene glycol (PG) on metabolic variables related to ovarian function and on oocyte developmental competence. Starting on Day 7 postpartum, each animal received an oral dose (500 ml) of either PG or water once daily. Blood samples were collected on Days 5, 15, 25 and 35 pp to measure insulin, non-esterified fatty acids (NEFAs), beta-hydroxybutyrate (BHB) and glucose concentrations. Oocytes were recovered by ultrasound guided follicular aspiration starting on approximately Day 30 postpartum and submitted to in vitro fertilization. Ovarian follicular activity was examined daily by ultrasonography from Day 7 until ovulation or Days 35-40 postpartum. Animals receiving PG had elevated insulin concentrations over the subsequent 90 min following dosing (P<0.05) compared to control animals. Glucose concentrations followed a similar pattern. Irrespective of treatment, concentrations of NEFA declined significantly from Days 15 to 35 postpartum. Administration of PG resulted in a decrease in NEFA (P<0.001) and BHB (P<0.001) over the subsequent 90 min compared to control animals. Treatment with PG had no effect on follicular dynamics, mean days to emergence of the first cohort of follicles postpartum, or days to dominance and duration of dominance for any follicular wave recorded postpartum. There was also no difference in mean days to first ovulation or in size of the preovulatory follicle between treatments. Oocyte quality as measured by blastocyst development after IVF was not affected by treatment. These results suggest that administration of PG has the ability to positively alter the systemic concentrations of a number of metabolic variables which have been related to fertility. However, we did not observe an effect of PG treatment on follicular dynamics or the length of the postpartum interval. An effect on oocyte developmental competence remains to be proven.     

Developmental competence of oocytes from prepubertal Bos indicus crossbred cattle

The aim of the present study was to evaluate the effect of age on embryogenic competence of oocytes recovered from Bos indicus crossbred calves and heifers. Cumulus-oocyte complexes (COCs) were collected from 4- to 7-month-old calves (experiment 1) and from 9- to 14-month-old heifers (experiment 2) during processing at an abattoir. In both experiments cow COCs were used as control. COCs were in vitro matured and fertilized, and the presumptive zygotes co-cultured with cumulus cells until 224 h post insemination (hpi). In experiment 1, the development rate during the first 68-72 hpi was similar (P > 0.05) between embryos derived from calves and cows. Fewer embryos from calves developed to the blastocyst stage, resulting in a lesser blastocyst production as well as lesser hatching rate (P < 0.05). The embryo development after blastocyst stage was, nevertheless, similar (P > 0.05) between blastocysts derived from calves and cows, suggesting that the development after blastocoele formation is not compromised in embryos derived from calves. In experiment 2, there were no differences (P > 0.05) on cleavage, blastocyst and hatching rates between embryos derived from prepubertal heifers and cows. The rate of blastocyst development until hatching was also similar (P > 0.05). These results indicate that oocytes from 9- to 14-month-old B. indicus crossbred heifers have the same developmental competence as oocytes derived from cows, while ocytes derived from 4- to 7-month-old B. indicus crossbred calves are less competent in developing to the blastocyst stage in vitro. It suggests that oocyte competence in B. indicus crossbred cattle is achieved around 9-14 months of age.     

Oxygen and steroid concentrations in preovulatory follicles of lactating dairy cows exposed to acute heat stress

Maternal heat stress reduces oocyte competence for fertilization and post-fertilization development, but the mechanism is unknown. The present experiment investigated two potential mechanisms: (1) reduced oxygen delivery to the preovulatory follicle (due to increased thermoregulatory vascular perfusion of skin and respiratory tract); (2) reduced follicular steroid synthesis. These hypotheses were tested by measuring the fractional concentration of oxygen and concentrations of estradiol-17beta and progesterone in follicular fluid of the preovulatory follicle of lactating Holstein cows. Estrous cycles were synchronized using GnRH on Day -9 and PGF(2alpha) on Day -2. On Day 0, all cows without a CL and with a large preovulatory follicle were assigned to control or heat stress treatments for 1d (beginning at 1030 h). Between 4 and 6 h after treatment (1430-1630 h), follicular fluid was aspirated by transvaginal puncture, and fractional oxygen concentration in follicular fluid of the dominant follicle was determined with a fluorometric fiber-optic oxygen sensor. There was no significant effect of heat stress on follicular fluid P(O2) or concentrations of estradiol-17beta or progesterone among cows that had follicular fluid steroid concentrations considered typical of a preovulatory follicle. Follicular oxygen concentration was 6.9+/-0.4% for control cows and 7.3+/-0.3% for heat-stressed cows. Oxygen concentration tended to be inversely correlated to follicular diameter (P=0.09). In conclusion, it was unlikely that reduced oocyte competence due to acute heat stress was caused by reductions in follicular concentrations of oxygen, estradiol-17beta, or progesterone.     

Influence of the breed of bull (Bos taurus indicus vs. Bos taurus taurus) and the breed of cow (Bos taurus indicus, Bos taurus taurus and crossbred) on the resistance of bovine embryos to heat

In vitro studies have shown that Bos taurus indicus (B. t. indicus) embryos submitted to heat shock at early stages of development are better able to survive as compared to Bos taurus taurus embryos. Embryo genotype influences resistance to heat shock thus leading to the question as to whether embryos sired by thermo-tolerant breeds exhibit the same resistance to heat shock. In the present study the influence of both oocyte and semen, on the resistance to heat shock (HS) at early stages of in vitro development, was assessed in B. t. indicus [Nelore (N) breed], B. t. taurus [Holstein (H) and Angus (A) breeds] and crossbreds. In Experiment 1, Nelore and crossbred oocytes were collected from slaughterhouse ovaries and fertilized with spermatozoa from Nelore and Angus bulls. Presumptive embryos were collected and randomly assigned to control (39 degrees C) or HS at 12, 48 or 96 h post insemination (hpi; 41 degrees C for 12h) treatments. The cleavage rates and proportion of embryos developing to the blastocyst and hatched blastocyst stages were recorded on Days 2, 8 and 10, respectively. Heat shock treatment decreased development of both Nelore and crossbred embryos. There was a significant interaction between time (12, 48 or 96 hpi) and temperature for blastocyst rates, i.e., the embryos became more thermotolerant as development proceeded. In Experiment 2, oocytes from Nelore and Holstein cows were fertilized with semen from bulls of either Nelore or Angus breeds, and subjected to 12 h HS at 96 hpi. Heat shock at 96 hpi, decreased embryo development. Additionally, cowxtreatment and bullxtreatment interactions were significant for blastocyst rates, i.e., both breed of cow and breed of bull affected the decline in blastocyst rate caused by heat shock treatment. In conclusion, the present results indicate that Nelore embryos (indicus) are more resistant to heat shock than Holstein (taurus) at early stages of in vitro development, and that embryos become more thermo-tolerant as development proceeds. Additionally, the resistance to heat shock was a result of the genetic contribution from both oocyte and spermatozoa.     

Comparison of oocyte developmental competence and follicular steroid content of nulliparous heifers and cows at different stages of lactation

Reduced reproductive performance and lower conception rates of lactating cows are closely associated with genetic progress for high milk production. In contrast, the fertility of nulliparous Holstein heifers has remained fairly stable over the years and appears to be markedly higher than that of mature lactating cows. Possible differences in oocyte quality and follicular steroid levels, which could be associated with the low fertility of high-lactating cows, were examined in 13-month-old heifers, cows around the time of first AI (60-95 d post-partum, yielding 49+/-2.4 kg/d) and cows at mid-lactation (120-225 d post-partum, yielding 37+/-2.1 kg/d). Estrus was synchronized by two doses of PGF2alpha and follicles (5-8 mm) were aspirated on days 4, 8, 11 and 15 of the cycle by an ultrasound-guided procedure. Oocytes were morphologically examined, matured in vitro, chemically activated and cultured for 8d. Cleavage rate and the proportion of developing parthenogenetic blastocysts were determined on days 3 and 8 post-activation, respectively. On day 17, heifers and cows received additional PGF2alpha and follicular fluids from preovulatory follicles were collected on day 19 perior to the expected estrus. Follicular-fluid volumes were similar in cows and heifers, as were estradiol, progesterone and androstenedione concentrations in the follicular fluid. Percentages of high-grade oocytes, proportions of cleaved oocytes and developed blastocysts did not differ between the groups. Results suggest that the fertility gap between nulliparous heifers and high-lactating cows is not directly related to steroid content in the preovulatory follicular fluid or oocyte developmental competence.     

Follicular dynamics during postpartum anestrus and the first estrous cycle in suckled or non-suckled Brahman (Bos indicus) cows

Brahman (Bos indicus) cows, were selected at 28+/-10 days after calving and analyzed by real time rectal ultrasonography three times a week, in order to evaluate and compare follicular and corpus luteum development during postpartum (PP) anestrus and the first PP estrous cycle under sylvopastoril conditions. Suckling (S, n=11) or non-suckling (NS, n=5) cows were evaluated in a zone of tropical dry forest (450m of altitude, mean temperature=27 degrees C, annual rainfall=1000mm). Estrous detection was performed twice daily by direct observation. Progesterone was quantified using RIA. From 28+/-10 days postcalving to resumption of estrous cycles, there were no differences (P>0.05) between NS and S cows for diameter of the dominant or first subordinate follicle, follicular growth rate, or interdominance interval. Silent ovulation, corpus luteum formation and subsequent progesterone concentrations ranging from 0.3 to 9. 7ng/ml, were found in both groups. The first calving to ovulation and calving to standing estrus intervals were shorter (P<0.01) in NS (34.8+/-5.81 and 41.2+/-9.03 days) than in S (65+/-4.82 and 81+/-6. 21 days) cows. Follicular development and progesterone concentrations during the first PP estrous cycle did not differ (P>0. 05) between NS and S cows. These results suggest that Brahman cows could have an early PP resumption of follicular recruitment if fed under sylvopastoril system conditions. However, non-suckled cows did have an earlier standing estrus and ovulation than did suckled cows.     

Effect of different stages of the follicular wave on in vitro developmental competence of bovine oocytes

This study aimed to investigate the developmental competence of ovum pick-up collected oocytes on three stages of the follicular wave: Days 2, 5 and 8. A group of 11 cows was used in successive cycles to perform ovum pick-up on either Day 2, 5 or 8 of an induced follicular wave (three sessions per stage). Follicular waves were initiated by puncturing the dominant follicle and all other follicles sized > or = 5 mm at Days 5-7 of the cycle. The plasma progesterone concentrations did not differ between the days of ovum pick-up: 4.0 +/- 1.8, 5.1 +/- 1.6 and 5.2 +/- 1.7 ng/ml for Days 2, 5 and 8, respectively. The proportion of oocytes with three or more layers of non-expanded cumulus cells was higher for Day 5 than Day 8, while Days 2 and 5 did not significantly differ from each other (85, 96 and 68% of 113, 60 and 101 oocytes for Days 2, 5 and 8, respectively). The proportion of oocytes competent to develop a blastocyst in an in vitro production system was higher for Days 2 and 5 than for Day 8: 27, 29 and 15% for the oocytes with fair to good cumulus investment and 23, 27 and 11%, respectively, when all oocytes were taken in account. This indicates that the dominant follicle reduces the developmental competence of oocytes from subordinate follicles at a relatively late stage of dominance. This finding has practical consequences for the handling of cows that undergo ovum pick-up only once or very irregularly. The embryo yield can then be improved by performing the ovum pick-up at Days 2-5 of the cycle or 2-5 days after ablation of the large follicles.     

Bovine follicular development and its effect on the in vitro competence of oocytes

The current knowledge is reviewed concerning correlations between follicular development in the cow and the competence of matured oocytes to develop into an embryo following IVF and IVC. At the follicular size of 3 mm, some oocytes become competent and the proportion of competent oocytes does not increase during development up to 7 mm. The proportion of competent oocytes increases greatly in follicles > 8 mm in both untreated and gonadotropin-stimulated cows. The competence of in vitro-matured oocytes from these large follicles is lower than the competence of in vivo-matured oocytes. These observations lead to the following concept. Oocytes have acquired an intrinsic capacity to develop into an embryo after IVM-IVF-IVC at the follicular stage of 3 mm, but require an additional "prematuration" to express this competence. In vivo, this prematuration occurs during preovulatory development before the occurrence of the LH surge. In follicles of 3-7 mm, a low level of atresia appears to improve the in vitro competence of oocytes which may act via a prematuration-like effect. A thorough understanding, however, of the effect of atresia and other factors on the competence of this highly heterogeneous oocyte population is still missing. Two routes to improve the embryo yield in ovum pick-up (OPU) practice are discussed.     

Changes in the cumulus-oocyte complex of subordinate follicles relative to follicular wave status in cattle

We investigated factors that affect cumulus-oocyte complex (COC) morphology and oocyte developmental competence in subordinate follicles on different days after follicular wave emergence in beef heifers. In Experiment 1, heifers (n = 13) were assigned at random to COC aspiration during the growing/static (Days 1 to 3) or regressing (Day 5) phase of subordinate follicle development (follicular wave emergence = Day 0). Follicular wave emergence was induced by transvaginal ultrasound-guided follicular ablation, ovaries were collected at slaughter, all follicles > or = 2 mm except the dominant follicle were aspirated, and COC were microscopically evaluated for morphology. There was a greater percentage of COC with expanded cumulus layers on Day 5 (42.4%) than on Days 1 to 3 (2.2%). In Experiment 2, heifers (n = 64) at random stages of the estrous cycle had all follicles > or = 5 mm ablated and 4 d later, 2 doses of PGF were injected 12 h apart; heifers were monitored daily by ultrasonography for ovulation (Day 0 = follicular wave emergence). Heifers were assigned to the following time periods for oocyte collection from subordinate follicles: Days 0 and 1 (growing phase), Days 2, 3 and 4 (static phase), and Days 5 and 6 (regressing phase). Ovaries were individually collected at slaughter, and all follicles > or 2 mm except for the dominant follicle were aspirated. The COC were morphologically evaluated and then matured, fertilized and cultured in vitro. Expanded COC were more frequent during the regressing phase (53.4%) than the growing or static phase (14.4 and 17.8%, respectively; P < 0.05). While the proportions of COC with > or = 4 layers of cumulus cells and denuded oocytes were higher (P < 0.05) in the growing and static phases, the production of morulae was highest (P < 0.05) with COC collected from subordinate follicles during the regressing phase. In Experiment 3, heifers (n = 18) were assigned at random to oocyte collection from subordinate follicles 3 and 4 d (static phase) or 5 and 6 d (regressing phase) after follicular wave emergence. The heifers were monitored ultrasonically for ovulation (Day 0 = follicular wave emergence); COC were collected from all follicles (> or = 5 mm) except for the dominant follicle by transvaginal ultrasound-guided follicle aspiration 3 to 6 d later. Recovered oocytes were stained and examined microscopically to evaluate nuclear maturation. A higher proportion of oocytes collected on Days 5 and 6 showed evidence of nuclear maturation (50%) than on Days 3 and 4 (8.3%; P < 0.05). Results support the hypothesis that COC morphology and oocyte developmental competence change during the growing, static and regressing phases of subordinate follicle development.     

Large-scale in vitro embryo production and pregnancy rates from Bos taurus, Bos indicus, and indicus-taurus dairy cows using sexed sperm

Herein we describe a large-scale commercial program for in vitro production of embryos from dairy Bos taurus, Bos indicus, and indicus-taurus donors, using sexed sperm. From 5,407 OPU, we compared the number of recovered oocytes (n = 90,086), viable oocytes (n = 64,826), and embryos produced in vitro from Gir (Bos indicus, n = 617), Holstein (Bos taurus, n = 180), 1/4 Holstein × 3/4 Gir (n = 44), and 1/2 Holstein-Gir (n = 37) crossbred cows, and the pregnancy rate of recipient cows. Viable oocytes were in vitro matured (24 h at 38.8 °C, 5% CO₂ in air) and fertilized by incubating them for 18 to 20 h with frozen-thawed sexed sperm (X-chromosome bearing) from Gir (n = 8) or Holstein (n = 7) sires (2 × 10⁶ sperm/dose). Embryos were cultured in similar conditions of temperature and atmosphere as for IVM, with variable intervals of culture (between Days 2 and 5) completed in a portable incubator. All embryos were transferred fresh, after 24 to 72 h of transportation (up to 2,000 km). On average, 16.7 ± 6.3 oocytes (mean ± SEM) were obtained per OPU procedure and 72.0% were considered viable. Total and viable oocytes per OPU procedure were 17.1 ± 4.5 and 12.1 ± 3.9 for Gir cows, 11.4 ± 3.9 and 8.0 ± 2.7 for Holstein cows, 20.4 ± 5.8 and 16.8 ± 5.0 for 1/4 Holstein × 3/4 Gir, and 31.4 ± 5.6 and 24.3 ± 4.7 for 1/2 Holstein-Gir crossbred females (P < 0.01). The mean number of embryos produced by OPU/IVF and the pregnancy rates were 3.2 (12,243/ 3,778) and 40% for Gir cows, 2.1 (2,426/1,138) and 36% for Holstein cows, 3.9 (1,033/267) and 37% for 1/4 Holstein × 3/4 Gir, and 5.5 (1,222/224), and 37% for 1/2 Holstein-Gir. In conclusion, we compared oocyte yield from two levels of indicus-taurus breeds and demonstrated the efficiency of sexed sperm for in vitro embryo production. Culturing embryos during long distance transportation was successful, with potential for international movement of embryos.     

Nutritional management of the donor cow

Nutrition of the donor cow can influence oocyte and embryo quality, which can affect the success of embryo transfer. Severe undernutrition compromised ovarian follicular development, with implications for superovulatory response and embryo quality. In postpartum lactating cows, undernutrition or inability to consume sufficient nutrients delayed resumption of ovulation, reduced the number of follicles, and compromised oocyte quality. Moderate undernutrition of nonlactating cows was unlikely to affect embryo quality; conversely, nonlactating animals on maintenance diets usually had better superovulatory responses and improved oocyte competence and embryo quality. The negative effects of overfeeding are thought to be mediated by alterations in endocrine cues, such as hyperinsulinemia and increased glucose and IGF-I, which may interfere with glucose transport in the embryo and increase apoptosis. Manipulating energy sources such as carbohydrates and fatty acids (FA) may influence embryo viability, but the effects of FA were not consistent in vitro; increasing concentrations of unsaturated FA in follicular and embryonic cells usually improved embryo viability and resistance to cryopreservation. Excess protein intake and increased urea and ammonia in body fluids can be toxic to embryos, impairing their development; these effects seemed to be associated with alterations in uterine pH and granulosa cell function. Likewise, toxins in feeds (e.g. gossypol), reduced embryo development and increased pregnancy losses. Diet of donor cows should be formulated to optimize the supply of nutrients to meet needs; however, manipulating energy intake, source of FA and protein content of donor diets, particularly moderate underfeeding in nonlactating cows, may further optimize responses.     

Long-term follicular dynamics and biochemical characteristics of dominant follicles in dairy cows subjected to acute heat stress

The objective of this study was to examine the quality of successive dominant follicles (DFs) after induced heat stress. Non-lactating dairy cows expressing estrus at normal intervals were allocated randomly to heat stress (HS; n=8) and control (C; n=8) groups. Cows received GnRH (100 microg, i.m.) on Day 0, a progesterone CIDR-B device on Day 4 and prostaglandin (PGF(2alpha); 25mg, i.m.) on Day 7 upon removal of the CIDR device. The DF and follicles >5mm were aspirated on Day 8, and GnRH (100 microg) injected following aspiration, to initiate a new follicular wave. In this manner, a DF was aspirated every 8 days (one "follicular cycle") for 10 cycles. After the first follicular cycle, HS cows were placed in environmental chambers for 7 days during the second follicular cycle (8h per day at 43.3 degrees C set point and 16h per day at 24 degrees C for 4 days, and 8h per day at 43.3 degrees C set point and 16h per day at 32.2 degrees C set point for 3 days; relative humidity, 40%) and thereafter maintained outdoors with control cows at a mean ambient temperature (18.5 degrees C; range 12.7-26 degrees C). Rectal temperature increased (P<0.001) in HS as compared with C cows (39.28+/-0.01 degrees C versus 38.78+/-0.01 degrees C). Concentrations of estradiol (E(2); 1662+/-189 versus 1493+/-188ng/ml) and progesterone (P(4); 44.7+/-5 versus 54.1+/-5.1ng/ml) in follicular fluid (FF) of DF did not differ between C and HS treatments, respectively. Total FF protein concentration was greater (P<0.05) in HS (99.7+/-2.3mg/ml) than in C (92.7+/-2.3mg/ml). Heat shock protein 90 (Hsp 90) in FF was not altered by heat stress. IGF-II ligand blots were conducted with FF samples (n=79) from four HS and four C cows. There was a predominance of IGFBP-3 in 76 of 79 FF samples, indicating healthy follicular status, and only three FF samples had the lower molecular weight IGFBP-2 indicative of a poor quality follicle. Plasma P(4) and E(2) concentrations did not differ between C and HS groups. The number of class 1 and 3 follicles increased during and just after heat stress, but the number of class 2 follicles did not differ between C and HS cows. Heat stress appeared to induce a decrease in follicular dominance, but GnRH-induced follicular cycles resulted in development of healthy preovulatory follicles in both groups.     

Use of group-specific antibodies to detect fecal progesterone metabolites during the estrous cycle of cows

Progesterone is metabolized to pregnanediones and hydroxylated pregnanes prior to its fecal excretion. Therefore, use of progesterone antibodies underestimates the actual amount of fecal metabolites. To improve the methodology of noninvasive fecal progesterone metabolite analysis, enzymeimmunoassays (EIA) using group-specific antibodies against 5-reduced 20-oxo-pregnane-C3-conjugates were developed. Fecal and milk samples were collected at 1- to 2-d intervals during the morning and evening milking throughout 1 estrous cycle in dairy cows (n = 12). Six immunoreactive metabolites were detected in the feces with high performance liquid chromatography (HPLC), eluting as 5α- and 5β-reduced pregnanes containing a 20-oxo-group (20-oxo-pregnanes). Fecal samples of 3 cows were analyzed by 3 EIAs using antibodies against 4-pregnene-6α-ol-3,20-dione 6HS:BSA (6HS-progesterone), 5α-pregnane-3β-ol-20-one 3HS:BSA and 5β-pregnane-3β-ol-20-one 3HS:BSA, respectively. The follicular and luteal phases were identifiable with each EIA. Luteal phase values and the differences between mean follicular (Days 0 to 2 and 19 to 21) and luteal phase (Days 10 to 16) values obtained with the 5-pregnane EIAs were 3- to 4-fold higher than with the 6HS-progesterone EIA. Since results with the former 2 EIAs were almost identical, the remaining samples were only analyzed by the EIA for 5β-pregnane-3α-ol-20-one. Fecal 20-oxo-pregnane concentrations were parallel to milk progesterone values, but had a lag time of about 0.5 d; the coefficient of correlation (P < 0.001) was 0.76 (y = 155.2 × + 37.2). Fecal 20-oxo-pregnane concentrations during the follicular and luteal phase were 39.5 ± 2.2 and 341 ± 15.2 ng/g feces, respectively. In conclusion, fecal 20-oxo-pregnanes are significantly correlated to milk progesterone concentrations. They consist of several metabolites and compared to a 6HS-progesterone antibody, their evaluation was improved using antibodies against 5-reduced pregnanes.     

Embryo recovery and pregnancy rates after the delay of ovulation and fixed time insemination in superstimulated beef cows

The aim of this study was to evaluate the effect of delaying ovulation subsequent to superstimulation of follicular growth in beef cows (Bos indicus) on embryo recovery rates and the capacity of embryos to establish pregnancies. Ovulation was delayed by three treatments using either progesterone (CIDR-B) or a GnRH agonist (deslorelin). Multiparous Nelore cows (n = 24) received three of four superstimulation treatments in an incomplete block design (n = 18 per group). Cows in Groups CTRL, P48 and P60 were treated with a CIDR-B device plus estradiol benzoate (EB, 4 mg, i.m.) on Day-5, while cows in Group D60 were implanted with deslorelin on Day-7. Cows were superstimulated with FSH (Folltropin-V, 200 mg), from Day 0 to 3, using twice daily injections in decreasing amounts. All cows were treated with a luteolytic dose of prostaglandin on Day 2 (08:00 h). CIDR-B devices were removed as follows: Group CTRL, Day 2 (20:00 h); Group P48, Day 4 (08:00 h); Group P60, Day 4 (20:00 h). Cows in Group CTRL were inseminated at 10, 20 and 30 h after first detected estrus. Ovulation was induced for cows in Group P48 (Day 4, 08:00 h) and Groups P60 and D60 (Day 4, 20:00 h) by injection of LH (Lutropin, 25 mg, i.m.), and these cows were inseminated 10 and 20 h after treatment with LH. Embryos were recovered on Days 11 or 12, graded and transferred to synchronized recipients. Pregnancies were determined by ultrasonography around Day 100. Data were analyzed by mixed procedure, Kruskal-Wallis and Chi-square tests. The number of ova/embryos, transferable embryos (mean +/- SEM) and pregnancy rates (%) were as follows, respectively: Group CTRL (10.8+/-1.8, 6.1+/-1.3, 51.5), P48 (12.6+/-1.9, 7.1+/-1.0, 52.3), P60 (10.5+/-1.6, 5.7+/-1.3, 40.0) and D60 (10.3+/-1.7, 5.0+/-1.2, 50.0). There were no significant differences among the groups (P > 0.05). It was concluded that fixed time AI in association with induced ovulation did not influence embryo recovery. Furthermore, pregnancy rates in embryos recovered from cows with delayed ovulation were similar to those in embryos obtained from cows treated with a conventional superstimulation protocol.     

Effect of treatment with progesterone and oestradiol when starting treatment with an intravaginal progesterone releasing insert on ovarian follicular development and hormonal concentrations in Holstein cows

Ovarian follicular development and concentrations of gonadotrophin and steroid hormones were studied in non-lactating Holstein cows following administration of progesterone (P(4)) or oestradiol benzoate (ODB) at the start of treatment with an intravaginal progesterone releasing insert (IVP(4)) in a 2 by 2 factorial experiment. Cows were treated at random stages of the oestrous cycle with an IVP(4) device (Day 0) and either no other treatment (n=8), 200 mg of P(4) IM (n=9), 2.0 mg of ODB IM (n=8) or both P(4) and ODB (n=9). Seven days later devices were removed and PGF(2alpha) was administered. Twenty-four hours later 1.0mg of ODB was administered IM. Oestrus was detected in 97.1% and ovulation in 64.7% (effect of treatment, P=0.41) of cows within 96 h of removing inserts. In the cows that ovulated, day of emergence of the ovulatory follicle was delayed (P<0.01) and more precise (P<0.05) in cows treated with ODB compared to the cows treated with P(4). Interval from wave emergence to ovulation and the diameter of the ovulatory follicle was less (P<0.05) in cows treated with ODB compared to cows treated with P(4). Combined treatment with P(4) and ODB at the time of starting treatment with an IVP(4) device did not significantly change the pattern of ovarian follicular development compared to treatment with ODB alone. Concentrations of LH and FSH in plasma were less in cows treated with ODB between Days 0 and 4 (P<0.05) while treatment with P(4) increased concentrations of FSH in plasma between Days 0 and 4 (P<0.05). When anovulatory cows were compared to ovulatory cows, diameters of follicles (P<0.001) and growth rate of follicles (P<0.01) were less in anovulatory cows between Days 7 and 9, while concentrations of FSH in plasma were greater (P<0.01), concentrations of LH similar (P>0.90) and concentrations of oestradiol were less (P=0.01) in the anovulatory cows between Days 4 and 10. Our findings support a hypothesis that ovarian follicular development following administration of P(4) or ODB at the start of treatment with an IVP(4) device differs. Anovulatory oestrus may have been associated with reduced maturity and/or later emergence of ovarian follicles.     

Utilization of the growth phase of the first follicular wave for bovine oocyte collection improves blastocyst production

Characteristics of the follicle population and oocyte developmental competence at selected stages of follicular development were studied in cows with the aim to increase embryo production derived from oocytes collected by transvaginal aspiration. In Experiment 1, the growth phase before dominant follicle selection and the low dominant phase during dominant follicle regression were compared. Twenty-four cyclic Holstein cows, 4 to 6 yr of age, were divided into 2 groups. Animals were synchronized using two injections of prostaglandin F2alpha at 11 d intervals, and onset of estrus was determined (Day 0). Using ultrasonography, all follicles were counted and classified. Oocytes were aspirated once on Days I through 3 (Group 1, n=5) or Days 15 and 16 (Group 2, n=3) of the estrus cycle. The experiment was carried out in 3 replicates. In Experiment 2, the growth phase of the first follicular wave before dominant follicle selection was characterized in detail. Twelve cows of the same breed and age were divided into 3 groups. Their first estrus was synchronized as in Experiment 1, and each following estrus was induced using one injection of prostaglandin F2alpha administered 4 to 6 d after each aspiration performed. The ovaries were examined, and oocytes were collected repeatedly (total of 5 times per cow) on Days 1 (Group 3, n=4), 2 (Group 4, n=4) or 3 (Group 5, n=4) after estrus at 10 d intervals during a 40 d period. Viable oocytes were matured, fertilized and cultured using the standard methods. In Experiment 1, the mean numbers (+/-SD) of all follicles and of recovered and viable oocytes per donor were higher in Group 1 than in Group 2, but only the mean numbers (+/-SD) of larger follicles and recovered oocytes were statistically significant (8.0 +/- 0.6 and 6.2 +/- 0.6.vs. 3.3 +/- 0.5 and 2.8 +/- 0.2; P< 0.05). In Experiment 2, the percentage of larger follicles out of all visible follicles and the mean numbers (+/-SD) of larger follicles per donor were significantly higher (P<0.05) in Groups 4 (75.7 and 9.1 +/- 2.7) and 5 (66.3 and 8.5 +/- 2.9) when compared to Group 3 (27.9 and 3.8 +/- 0.8). The development rate of fertilized oocytes was significantly higher (P<0.05) in Groups 4 (27.8) and 5 (27.5) than in Group 3 (12.8). It can be concluded that it is possible to improve the efficiency of transvaginal aspiration and in vitro embryo production by utilization of the growth phase of the first follicular wave before dominant follicle selection.     

Relationship between concentrations of immunoreactive insulin-like growth factor-I in follicular fluid and various biochemical markers of differentiation in bovine antral follicles

Three experiments were conducted to determine the relationship between concentrations of insulin-like growth factor-I (IGF-I) in ovarian follicular fluid and various biochemical markers of follicular differentiation in bovine follicles. In Experiment I, ovaries were removed on Days 7, 14, 28, 42, or 56 after parturition from a total of 21 cows. In Experiment II, ovaries of 31 cows were removed between Days 20 and 30 postpartum after 48 or 96 h of either saline (0.9% NaCl, 5 ml) or luteinizing hormone-releasing hormone (LHRH, 500 ng/5 ml saline) injections given every 2 h via jugular cannulae. In Experiment III, ovaries of six cows were removed 48-50 h after a 35-mg injection of prostaglandin F2 alpha during the midluteal phase of an estrous cycle. In Experiments I and II, all follicles greater than or equal to 8.0 mm in diameter were removed from each ovary (n = 33 and 46, respectively). In Experiment III, fluid from all follicles greater than 4 mm in diameter were removed individually (n = 10), and fluid from follicles 1-4 mm in diameter were pooled for each cow. Follicles for each experiment were further categorized as either estrogen-active (E-A, concentration of estradiol greater than progesterone in follicular fluid) or estrogen-inactive (E-I, concentration of progesterone greater than estradiol in follicular fluid). Measurements of immunoreactive IGF-I (i-IGF-I) were made after separating IGFs from their binding proteins with an acid-ethanol extraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of the dominant follicle on oocytes from subordinate follicles

As the oocyte grows within the follicle, a number of factors influence its health and developmental competence. These factors include follicle size, day of estrous cycle, level of atresia and influence of other follicles such as the dominant follicle. Follicles were dissected from ovaries of synchronized dairy cows on four days during the estrous cycle, and the oocyte from each follicle collected, matured, fertilized and cultured singly until Day 8. Development to blastocyst was greater in oocytes collected during phases of follicular growth than those collected during phases of follicular dominance (P<0.001) over all follicle size categories. Oocyte competence tended to increase with increasing follicle size (P<0.1). Follicular cells analyzed by flow cytometry showed an increase in proportion of apoptotic cells in subordinate follicles during the dominant phase compared to growth phase (P<0.05). Thus, the dominant follicle on both oocyte competence and levels of atresia. Further studies on the effect of dominance has shown that lactate production in cumulus-oocyte-complexes (COCs) from medium-sized follicles collected during a dominance phase and small follicles collected during a growth phase are no different from other follicles, despite having significantly lower uptake of glucose (P<0.1). Thus, COCs from different follicle subclasses differ in their nutrient requirements, and current IVM technology needs further improvement to better assist those oocytes that are developmentally challenged.