Transvaginal ultrasound guided follicular aspiration of bovine oocytes

A transvaginal ultrasound guided follicular aspiration technique was developed for the repeated collection of bovine oocytes from natural cycling cows. In addition, the feasibility of using this method for collecting immature oocytes for in vitro embryo production was also evaluated. Puncturing of visible follicles for ovum pick-up was performed in 21 cows over a three month period. All visible follicles larger than 3 mm were punctured and aspirated three times during the estrous cycle on Day 3 or 4, Day 9 or 10 and Day 15 or 16. The mean (+/- SEM) estrous cycle length after repeated follicle puncture was 22.2 +/- 0.3 days. The mean total number of punctured follicles per estrous cycle was 12.6 +/- 0.3. The largest (P<0.05) number of follicles punctured (5.1 +/- 0.3) for ovum pick-up was on Day 3 or 4 of the estrous cycle. The overall recovery rate of 541 punctured follicles was 55%. Most oocytes (P<0.05) were aspirated from follicles smaller than 10 mm. Following in vitro maturation and fertilization (IVM/IVF), 104 oocytes were transferred to sheep oviducts. Six days later, 75 ova/embryos were recovered, after flushing the oviduct of the sheep, of which 24% developed into transferable morulae and blastocysts. In this study, a reliable nonsurgical, follicular aspiration procedure was used for the repeated collection of immature oocytes which could be used successfully for in vitro production of embryos. This procedure offers a competitive alternative to conventional superovulation/embryo collection procedures.     

Repeated ovum pick-up in Italian Mediterranean buffalo cows

The potential of the ovum pick-up technique, used over a long period, was evaluated in 6 Italian Mediterranean buffalo cows that had more than 500 d open. The cows were submitted to ovum pick-up twice weekly for 2 mo. An additional 2-mo cycle of ovum pick-up was performed in 3 of the buffalo. The ovum pick-up sampling did not affect the resumption of reproductive activity of these animals. In fact, all the buffalo conceived, on average, 47.5 +/- 27.5 d after the last ovum pick-up. An average of 5.48 follicles was punctured, and 2.71 oocytes were collected per session. However, only 53.5% of these oocytes were suitable for in vitro embryo production. The number of punctured follicles differed between individual cows. There were no differences in the number of collected oocytes or in the recovery rates. The number of punctured follicles, the number of collected oocytes and the recovery rate were similar in the first and second months; the quality of the oocytes was, however, better in the second than in the first month (P < 0.05). The increasing interval between 2 consecutive ovum pick-up sampling (intersession interval) caused an increase of the percentage of large follicles. Moreover, the increase of the intersession interval from 4 to 5 d decreased the quality of the collected oocytes (P < 0.05). The efficiency of in vitro production of embryos to expanded blastocysts was 16.7%.     

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.     

Studies of FSH-P induced follicular growth in cows

Because cow ovaries do not contain a dominant follicle before Day 3 of the estrous cycle, we hypothesized that gonadotropin treatment early in the estrous cycle would induce growth of multiple follicles and could be used to induce superovulation. In Experiment 1, when 16 cows were treated with FSH-P beginning on Day 2 of the estrous cycle and were slaughtered on Day 5, all cows responded to gonadotropin treatment by exhibiting a large number ( approximately 19) of estrogenactive follicles >/= 6 mm. In Experiment 2, in response to FSH-P treatment from Day 2 to Day 7, and fenprostalene treatment on Day 6, 11 of 15 cows exhibited estrus and had a mean ovulation rate of 23.7 +/- 1.5. In Experiment 3, an FSH-P treatment regimen identical to that used in Experiment 2 was administered to cows beginning either on Day 2 (Day-2 cows; n=14) or Day 10 (Day-10 cows; n=11) of the estrous cycle. Twelve of 14 Day-2 cows and all Day-10 cows exhibited estrus after fenprostalene treatment. Day-2 cows exhibited 34.3 +/- 7.0 ovulations, which was less (P < 0.05) than that exhibited by Day-10 cows (48.3 +/- 4.4). However, the proportion of embryos recovered per corpus luteum was about 2-fold greater (P < 0.05) for Day-2 cows than for Day-10 cows (0.49 +/- 0.08 vs 0.27 +/- 0.06). These data indicate that beginning gonadotropin treatment early in the estrous cycle, when a dominant follicle is not present, provides an efficacious means to induce growth of multiple follicles and superovulation in cows. However, when FSH was administered for 6 d, beginning the treatment on Day 10 also resulted in a consistent and efficacious response.     

Ovarian superstimulation after follicular wave synchronization with GnRH at two different stages of the estrous cycle in cattle

The objective of this study was to evaluate superovulatory programs based on synchronization of follicular waves with GnRH at 2 different stages of the estrous cycle. Sixteen Holstein cows were randomly assigned to 1 of 3 groups and administered GnRH (Cystorelin, 4 ml i.m.) between Days 4 and 7 (Groups 1 and 3) or between Days 15 and 18 (Group 2) of the estrous cycle (estrus = Day 0). Four days after GnRH treatment, > or = 7-mm follicles were punctured in Groups 1 (n = 6) and 2 (n = 6) or were left intact in Group 3 (n = 4). All cows were superstimulated 2 d later (i.e., from Days 6 to 10 after GnRH treatment) with a total of 400 mg NIH-FSH (Folltropin-V) given twice daily in decreasing doses. The GnRH treatment caused a rapid disappearance of large follicles (P < 0.005), rapid decrease in estradiol concentrations (P < 0.003), and increase in the number of recruitable follicles (4 to 6 mm; P < 0.04), indicative of the emergence of a new follicular wave within 3 to 4 d of treatment. Between 4 and 6 d after GnRH treatment, the mean number of 4- to 6-mm follicles decreased (4.7 +/- 1.8 to 1.5 +/- 3.3) in the nonpunctured group but increased (3.9 +/- 1.0 to 7.3 +/- 1.9) in the punctured group of cows (P < 0.05). In response to FSH treatment, the increase in the number of > or = 7-mm follicles was delayed by approximately 2 d in the nonpunctured group (P < 0.006). Moreover, the mean number of > or = 7-mm follicles at estrus was higher (16.9 +/- 1.7 vs 11.5 +/- 3.0; P < 0.1) in the punctured than the nonpunctured group. The increase in progesterone concentration after estrus was delayed in the nonpunctured group (P < 0.1) compared with the punctured follicles. Mean numbers of CL as well as freezable (Grade 1 and 2) and transferable (Grade 1, 2 and 3) embryos were similar (P > 0.1) in punctured and nonpunctured groups. Spontaneous estrus did not occur prior to cloprostenol-induced luteolysis in any group, and stage of the estrous cycle during which GnRH was given did not affect (P > 0.1) hormonal and follicular responses in the punctured groups. In conclusion, GnRH given at different stages of the estrous cycle promotes the emergence of a follicular wave at a predictable time. Puncture of the newly formed dominant follicle increases the number of recruitable follicles (4 to 6 mm) 2 d later and, in response to superstimulation with FSH, causes a greater number and faster entry of recruitable follicles into larger classes (> or = 7 mm) and a faster postovulatory increase in progesterone concentrations.     

Follicular dynamics, repeatability and predictability of follicular recruitment in cows undergoing repeated follicular puncture

The aim of our study was to characterize follicular recruitment and growth in response to the repeated removal of follicles. All tertiary follicles (> 2 mm of diameter) in the ovaries of 10 non-lactating Holstein Friesian cows were punctured at midcycle (Day 0) by means of an ultrasound-guided needle. Puncture sessions were scheduled twice weekly at 3- or 4-d intervals over 3 mo. In the middle of the experiment, i.e., Week 7, the effects of 2- and 5-d intervals between follicular punctures were tested and compared with the previous 3- and 4-d intervals. After this period, 6 animals were slaughtered to study the effect of puncturing on gross ovarian morphology. The protocol of puncturing follicles with 3- to 4-d intervals was continued for an additional 3 mo in the remaining 4 animals. Twice-weekly puncturing of all tertiary follicles abolished estrous cycles and lead to an increase in follicular wave frequency without apparent negative effects on either the reproductive tract or ovaries. After puncture the new follicular wave attained full numerical development within 3 d. Two-day intervals resulted in a lower number of follicles than the 3-d interval (11.0 -/+ 3.8 vs 15.4 -/+ 6.1; P < 0.05). In contrast 4- and 5-d intervals between puncture resulted in an increase in follicle size when compared with that of the 3-d interval. The mean+SD number of recruited follicles varied between animals ranging between 78 +/- 2.5 to 19.2 -/+ 6.0. The mean number of follicles recruited increased from the first month (March) to the third month (May) of sampling (11.8 +/- 4.7 vs 16.4 +/- 6.5; P < 0.01), and then decreased between the third (May) and the sixth (August) month of sampling (21.5 +/- 4.7 vs 16.8 +/- 5.0; P < 0.01). During the experiment, the number of recruited follicles varied cyclically, with waves having a length of 6 puncture session (PS) or 3 wk (i.e., the mean length the bovine estrous cycle). Follicular recruitment after repeated ovum pick-up showed a high repeatability (r = 0.576) A model was also developed showing good predictability of the potential of animals to recruit follicles on the basis of the first 4 to 6 PS. Our results showed that despite large variation in follicular recruitment between animals, the high repeatability and good predictability of follicle recruitment demonstrates the possibility of characterizing animals on their potential for follicle growth.     

Inhibition of estrus and corpora lutea function with Norgestomet

Forty-five nonpregnant, nonlactating, Angus and Brangus cows were utilized to determine how long a Norgestomet ear implant would inhibit estrus when administered at various stages of an estrous cycle. All cows completed a nontreated estrous cycle to ensure normal cyclicity. At the second observed estrus (estrus = Day 1), cows were randomly allotted to be treated at metestrus (Day 3 or Day 4, n = 15); at diestrus (Day 9 or Day 10, n = 14); or at proestrus (Day 15 or Day 16, n = 16). All cows received a 2-ml intramuscular injection of 3 mg of Norgestomet accompanied by a 6-mg Norgestomet ear implant, which remained in situ for 21 days, or until individual cows were observed in estrus. Estrus was inhibited for a mean (+/- SEM) of 18.7 +/- 0.7, 19.9 +/- 0.8, and 17.0 +/- 0.8 days, respectively, when cows were treated at metestrus, diestrus, and proestrus (metestrus and diestrus vs proestrus; P < 0.05). Estrus was inhibited for an entire 21-day implantation period in 27, 50, and 38% of cows treated at metestrus, diestrus, and proestrus, respectively (P > 0.10). Norgestomet inhibited estrus in all cows for 11, 17, and 11 days after implantation when treatment was initiated at metestrus, diestrus, and proestrus, respectively (P > 0.10). These data indicate that a 6-mg Norgestomet ear implant effectively inhibits estrus in all cows for a maximum of 11 days, with some cows exhibiting estrus by Day 12 with the Norgestomet implant in situ.     

Development of dominant follicles and length of ovarian cycles in post-partum dairy cows

The resumption of ovarian activity after normal calvings was studied in 18 lactating Friesian cows. Since, in 17 cows, first post-partum ovulation occurred without overt oestrous behaviour being detected, the resultant cycles were called 'ovarian cycles'. The mean (+/- s.d.) length of the ovarian cycles was 21.0 +/- 8.7 days. The duration of cycles tended to be normal (18-24 days) or long (greater than or equal to 25 days) when the ovulatory dominant follicles were identified before Day 10 post partum; they were consistently short (9-13 days) when dominant follicles identified after Day 20 post partum ovulated. When such follicles were detected between Days 10 and 20 post partum, long, normal and short ovarian cycles were detected. The number of waves of follicular growth with associated dominant follicles observed during the ovarian cycles tended to be related to cycle length; short cycles had 1 dominant follicle, normal cycles predominantly 2, and long cycles mostly 3 dominant follicles. The mean (+/- s.d.) duration of 13 oestrous cycles studied was 23.1 +/- 2.1 days. Of these cycles, 7 had 3 and 6 had 2 dominant follicles. The oestrous cycles with 3 dominant follicles had a mean (+/- s.d.) duration of 24.0 +/- 1.2 days and the respective dominant non-ovulatory follicles reached maximum sizes on Days 8 and 18, respectively; oestrous cycles with 2 dominant follicles were 22.2 +/- 2.6 days in duration, and the dominant non-ovulatory follicle reached maximum size by Day 8. Ovarian follicular development during the first 45 days of pregnancy was characterized by the growth and regression of successive dominant follicles, each lasting 10-12 days. These results show that the first ovarian cycle was predominantly short when the ovulatory dominant follicle was first detected after Day 20 post partum.     

Effect of repeated eCG treatments and ovum pick-up on ovarian response and oocyte recovery during early pregnancy in suckling beef cows

This study was designed to evaluate in suckling early pregnant beef cows with and without eCG-pre-stimulation: (i) the influence of day gestation (from 40 to 101 days) and the consecutive eCG treatments on the follicular growth induced by means of ultrasound-guided transvaginal follicle ablation (FA; all follicles ≥ 5 mm) and the number and quality oocytes recovered by ovum pick-up (OPU) and (ii) the possible effects of repeated hormonal stimulation and FA/OPU on pregnancy outcome. Twelve suckling early pregnant Angus cows (40 days post fixed-time artificial insemination) were randomly assigned to each of two groups (n=6 group(-1)). Group 1 treatments included: FA (Day 0), eCG (1600 IU; Day 1) and OPU (Day 5). Group 2: as cited Group 1 with no eCG treatment. In both groups, OPU was repeated five times (Days 45, 59, 73, 87 and 101 of gestation). The numbers (mean ± SEM) of class II (5-9 mm; 4.3 ± 0.9) and class III (≥10 mm; 2.5 ± 0.4) follicles visualized per cow per OPU session in eCG-treated cows were greater (P<0.05) than for non-treated cows (0.9 ± 0.1 and 0.9 ± 0.1, respectively). In contrast, the number (mean ± SEM) of class I (<5mm) follicles per cow per OPU session was lower for cows with eCG treatment (2.8 ± 0.4) than for non-treated cows (5.7 ± 0.5). The mean number of aspirated follicles was not significantly different (P<0.05) between eCG-treated cows and non-treated cows at 45 and 59 days of pregnancy. However, the mean number of aspirated follicles was greater (P=0.03) in eCG-treated cows than non-treated cows from 73 day of pregnancy onwards. The numbers (mean ± SEM) of recovered oocytes and viable oocytes/cow/session were greater (P<0.05) for eCG-treated cows (2.2 ± 0.2 and 1.6 ± 0.4, respectively) than for non-treated cows (1.0 ± 0.2 and 0.9 ± 0.2, respectively). No donor pregnancies were lost either during or following OPU procedure. We can conclude that (1) eCG-treated pregnant suckled cows can be a source of oocytes for IVF at least to 100 days of gestation and (2) repeated FA/eCG treatment/OPU procedures did not affect the pregnancy outcome.     

Ultrasonographic determination of ovarian follicular. Development in superovulated heifers pretreated with FSH-P at the beginning of the estrous cycle

On Day 3 of the estrous cycle (estrus = Day 0), dairy heifers were given either 10 mg i.m. FSH-P (FSH-P primed; n = 9) or a saline vehicle (saline primed; n = 9). On Day 10, all heifers were superovulated with FSH-P (total = 27.7 mg i.m.) in declining doses over 5 d. Heifers were inseminated artificially at estrus. From Day 2 until estrus, the number and size of follicles >2 mm were monitored daily by ultrasonography. The mean (+/- SEM) number of corpora lutea (CL) (6.2 +/- 1.5 vs 10.7 +/- 0.9; P<0.05) and the mean number of recovered embryos and unfertilized ova (3.6 +/- 1.7 vs 8.4 +/- 2.2; P<0.05) were lower in FSH-P-primed than in saline-primed heifers. Prior to initiation of superovulation, follicles >10 mm appeared on Days 6 to 7 in saline-primed heifers but only on Days 8 to 10 in FSH-P-primed heifers (P<0.05). Also, until Day 10, the mean number of follicles 4 to 6 mm and 7 to 10 mm was higher (P<0.05) in FSH-P-primed than in saline-primed heifers. After initiation of the superovulatory treatment (Day 10 to estrus), saline-primed heifers had a greater and faster increase in the mean number of follicles >10 mm (P<0.02) than FSH-P-primed heifers did. Depletion in the number of follicles 2 to 3 mm (P<0.001) between Day 10 and estrus and in the number of follicles 4 to 6 mm (P<0.05) between Day 12 and estrus occurred in both groups of heifers. Decreased superovulatory response and embryo recovery in FSH-P-primed heifers may have been due to the presence of large follicles (>10 mm) prior to the initiation of the superovulatory treatment which reduced the ability of small follicles to grow into larger size classes during superovulatory treatment.     

Body condition influences maintenance of a persistent first wave dominant follicle in dairy cattle

The objective of this study was to determine whether plasma concentrations of progesterone (P4) from a controlled internal drug releasing (CIDR) device (approximately 2 ng/ml) were adequate to sustain a persistent first wave dominant follicle (FWDF) in low body condition (LBC, body condition score [BCS] 1 = lean, 5 = fat [2.3 +/- 0.72, n = 4]) compared with high body condition (HBC, BCS = 4.4 +/- 0.12, n = 4) nonlactating dairy cows. On Day 7 of the estrous cycle (Day 0 = estrus), cows were treated with PGF2 alpha (25 mg i.m. Lutalyse, P.M., and Day 8 A.M.) and a used CIDR device containing P4 (1.2 g) was inserted into the vagina until ovulation or Day 16. Plasma was collected for P4 and estradiol (E2) analyses from Day 5 to Day 18 (or ovulation), and ovarian follicles were monitored daily by ultrasonography. Mean concentrations of plasma P4 were greater in HBC than LBC cows between Days 5 and 7 (4.6 > 3.4 +/- 0.37 ng/ml; P < 0.04). All LBC cows maintained the first wave dominant follicle and ovulated after removal of the CIDR device (18.3 +/- 0.3 d, n = 3; Cow 4 lost the CIDR device on Day 11 and ovulated on Day 15), whereas in the HBC cows ovulation occurred during the period of CIDR exposure (11.3 +/- 0.3 d; n = 3; a fourth cow developed a luteinized first wave dominant follicle that did not ovulate during the experimental protocol on Day 19). Mean day of estrus was 17 +/- 0.4 for LBC (n = 3) and 10 +/- 0.4 for HBC (n = 3) cows. Sustained concentrations of plasma E2 (12.9 +/- 2.8 pg/ml; Days 8 to 17) in LBC cows reflected presence of an active persistent first wave dominant follicle. The differential effect of BCS on concentrations of plasma P4 (y = ng/ml) was reflected by the difference (P < 0.01) in regressions: yLBC = 19.9 - 3.49x + 0.166x2 vs yHBC = 37.3 - 7.04x + 0.340x2 (x = day of cycle, Days 7 to 12). Although P4 concentration was greater for HBC cows prior to Day 8, a greater clearance of plasma P4 released from the CIDR device in the absence of a CL altered follicular dynamics, leading to premature ovulation in the HBC cows. A greater basal concentration of P4 was sustained in LBC cows that permitted maintenance of a persistent first wave dominant follicle.     

Potential use of ovum pick-up for embryo production and breeding in cattle

The efficacy of transvaginal ultrasound-guided puncturing of ovarian follicles for collecting immature oocytes in cattle was studied. Three experiments were conducted to examine the effects of puncturing on follicle recruitment and on the number of oocytes collected. Puncture sessions were executed twice weekly at regular intervals of 3 and 4 d respectively. The oocytes were matured, fertilized and allowed to develop in vitro and the number of transferable embryos was recorded. The health of the cows was checked daily. In Experiment 1, dairy cows (n=10) were punctured over a period of 5 mo, and the collected oocytes were fertilized with the semen of 1 bull. In Experiment 2, oocytes were collected from one 12 year old high pedigree dairy cow and an one month pregnant cow were punctured. The oocytes of the old cow were fertilized with semen of 8 different bulls. In Experiment 3, beef cows (n=6) were punctured over a 2 mo period and the semen of 2 different bulls of the same breed was used to fertilize the oocytes from 3 of these cows. In Experiment 1, 14.5 +/- 0.4 (mean +/- SEM) follicles were punctured per session, and 8.0 +/- 0.3 (mean +/- SEM) oocytes were recovered. A mean of 16% of the oocytes developed into transferable embryos with a pregnancy rate of 40%. The results did not differ between the months of the experiments, indicating that the transvaginal puncturing method can be used successfully over a 5 mo period. No detrimental effects were observed after clinical and post mortem examinations, nor did breed, age or reproductive status appear to affect the results. However, large differences were observed between individual cows and between cow/bull combinations.     

Endocrine and ovarian responses associated with the first-wave dominant follicle in cattle.

To examine endocrine and biochemical differences between dominant and subordinate follicles and how the dominant follicle affects the hypothalamic-pituitary-ovarian axis in Holstein cows, the ovary bearing the dominant follicle was unilaterally removed on Day 5 (n = 8), 8 (n = 8), or 12 (n = 8) of synchronized estrous cycles. Follicular development was followed daily by ultrasonography from the day of detected estrus (Day 0) until 5 days after ovariectomy. Aromatase activity and steroid concentrations in first-wave dominant and subordinate follicles were measured. Intact dominant and subordinate follicles were cultured in 4 ml Minimum Essential Medium supplemented with 100 microCi 3H-leucine to evaluate de novo protein synthesis. Five days after unilateral ovariectomy, cows were resynchronized and the experiment was repeated. Follicular growth was characterized by the development of single large dominant follicles, which was associated with suppression of other follicles. Concentrations of estradiol-17 beta (E2) in follicular fluid and aromatase activity of follicular walls were higher in dominant follicles (438.9 +/- 45.5 ng/ml; 875.4 +/- 68.2 pg E2/follicle) compared to subordinate follicles (40.6 +/- 69.4 ng/ml; 99.4 +/- 104.2 pg E2/follicle). Aromatase activity in first-wave dominant follicles was higher at Days 5 (1147.1 +/- 118.1 pg E2/follicle) and 8 (1028.2 +/- 118.1 pg E2/follicle) compared to Day 12 (450.7 +/- 118.1 pg E2/follicle). Concentrations of E2 and androstenedione in first-wave dominant follicles were higher at Day 5 (983.2 +/- 78.2 and 89.5 +/- 15.7 ng/ml) compared to Days 8 (225.1 +/- 78.6 and 5.9 +/- 14.8 ng/ml) and 12 (108.5 +/- 78.6 and 13.0 +/- 14.8 ng/ml). Concentrations of progesterone in subordinate follicles increased linearly between Days 5 and 12 of the estrous cycle. Plasma concentrations of FSH increased from 17.9 +/- 1.4 to 32.5 +/- 1.4 ng/ml between 0 and 32 h following unilateral removal of the ovary with the first-wave dominant follicle. Increases in plasma FSH were associated with increased numbers of class 1 (3-4 mm) follicles in cows that were ovariectomized at Day 5 or 8 of the cycle. Unilateral ovariectomy had no effects on plasma concentrations of LH when a CL was present on the remaining ovary. First-wave dominant follicles incorporated more 3H-leucine into macromolecules and secreted high (90,000-120,000) and low (20,000-23,000) molecular weight proteins that were not as evident for subordinate follicles at Days 8 and 12.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of ultrasonically-guided follicle aspiration on estrous cycle and follicular dynamics in Holstein cows

Two experiments were conducted to test the effect of ultrasonically-guided follicle aspiration on estrous cycle and follicular dynamics in Holstein cows. The objective of the first experiment was to determine if aspiration of all visible follicles would influence the estrous cycle. All visible follicles > or = 5 mm in diameter were punctured and the oocytes aspirated once during the second and fourth cycle, with no aspirations occurring during Cycles 1, 3 and 5 in 4 Holstein cows. Between aspirations the follicles were scanned ultrasonically every 2 to 3 d to monitor follicular waves. Estrus was monitored twice daily and plasma progesterone concentrations were measured daily. The inter-estrus periods were significantly longer when aspiration was performed than in the 3 cycles in which aspiration did not occur (25.0 vs 21.1 d; P < 0.01). To determine the maximum number of follicles harvestable on a continuing basis, a second experiment was conducted to contrast unstimulated cows subjected to ultrasonically-guided oocyte collection twice-weekly with animals from which oocytes were collected once a week after gonadotrophin stimulation. All visible follicles of multiparous Holstein cows were aspirated every 3 or 4 d (unstimulated group, n = 4), and the total number of follicles was compared with the number of follicles in cows that were aspirated weekly after 400 mg FSH were administered over 2 d (stimulated, n = 6). The study continued for 8 consecutive weeks. The weekly mean number of follicles available for aspiration in the unstimulated cows was 15.7 +/- 3.3 (mean +/-SEM), which was not different from the 14.2 +/- 1.9 follicles available for aspiration from the FSH-stimulated cows. Within the unstimulated cows, there was an increased number of follicles available for aspiration over time during the period of study (P < 0.001). The number of follicles available for aspiration from the stimulated cows did not change, but plasma concentrations of progesterone increased over time as persistent luteal tissue developed on the ovaries.     

A comparison of a mechanical sector and a linear array transducer for ultrasound-guided transvaginal oocyte retrieval (OPU) in the cow

A comparison was made between a linear array and a mechanical multiple angle sector (MAP) transducer for ultrasound-guided transvaginal oocyte retrieval (ovum pick-up, OPU) in the cow. The ovaries of five dairy cows were punctured, in a twice-weekly OPU program lasting for 4 weeks, using two different 5.0-MHz transducers equipped with an identical disposable needle-guidance system. Both ovaries were visualized using each transducer before puncture and the number of follicles with a diameter of less than 5 mm (small) and with a diameter equal to or greater than 5 mm (large) was recorded. Subsequently, one ovary of the pair was punctured guided by the MAP, while the other was punctured using the linear array transducer. During the next puncture session on a given animal, the two systems were switched and used on the alternate ovary in a crossover design. Parameters assessed for each system were: the total number of follicles visualized in each diameter class, and the total number of retrieved oocytes per cow. A significant difference was found for the ability to visualize smaller follicles in favor of the MAP transducer, with an average visualization of 71.6 +/- 30.3 small follicles per cow during the 4-week trial period, compared to 59.8 +/- 25.7 for the linear array transducer (t-test for paired samples, P = 0.007). No differences were found in the visualization of large follicles. A numerically greater number of oocytes were retrieved using the MAP transducer, compared to the linear array, (averages of 14.2 +/- 7.2 versus 7.4 +/- 6.1, respectively), although these differences were not statistically significant. In conclusion, both systems can be effectively used for oocyte retrieval in the cow, however, the MAP transducer demonstrated superior visualization of small follicles.     

Follicular growth, ovulation and embryo recovery in dairy cows given FSH at the beginning or middle of the estrous cycle

Variability in the superovulation response is an important problem for the embryo transfer industry. The objective of this study was to determine whether FSH treatment at the beginning of the cycle would improve the ovulation rate and embryo yield in dairy cows. Twenty-eight postpartum cyclic dairy cows were allocated at random to 4 treatment groups (A, B, C and D). Group A cows (n = 10) received FSH (35 mg) at a decreasing dose, starting on Day 9 (Day 0 = day of estrus) for 5 days followed by PGF(2alpha) (35 mg) on Day 12. Cows assigned to Groups B, C and D (n = 6 cows each, respectively) were given 35 mg FSH at a decreasing dose from Days 2 to 6 followed by PGF(2alpha) on Day 7. Group C and D cows received PRID inserts from Day 3 to Day 7. Cows in Group D additionally received 1000 IU hCG 60 hours after PGF(2alpha) treatment. Ovaries were scanned daily using a real time ultrasound scanner from the beginning of FSH treatment until embryo recovery, to monitor follicular development, ovulation and the number of unovulated follicles. Embryos were recovered from the uterus by a nonsurgical flushing technique 7 days after breeding. There were no differences (P>0.01) in the number of follicles > 10 mm at 48 hours after PGF(2alpha) treatment among the 4 groups. The mean numbers of follicles were 10.6 +/- 1.2, 9.3 +/- 1.3, 12.2 +/- 1.3 and 15.0 +/- 2.9 for Groups A, B, C and D, respectively. A significantly (P<0.001) higher number of ovulations was observed and a larger number of embryos was recovered in Group A than in the other groups. The results of this study indicate that superovulation with FSH at the beginning of the cycle causes sufficient follicular development but results in very low ovulation and embryo recovery rates.     

The synchrony of prostaglandin-induced estrus in cows was reduced by pretreatment with hCG

The induction of optimal synchrony of estrus in cows requires synchronization of luteolysis and of the waves of follicular growth (follicular waves). The aim of this study was to determine whether hormonal treatments aimed at synchronizing follicular waves improved the synchrony of prostaglandin (PG)-induced estrus. In Experiment 1, cows were treated on Day 5 of the estrous cycle with saline in Group 1 (n = 25; 16 ml, i.v., 12 h apart), with hCG in Group 2 (n = 27; 3000 IU, i.v.), or with hCG and bovine follicular fluid (bFF) in Group 3 (n = 21; 16 ml, i.v., 12 h apart). On Day 12, all cows were treated with prostaglandin (PG; 500 micrograms cloprostenol, i.m.). In Experiment 2, cows were treated on Day 5 of the estrous cycle with saline (3 ml, i.m.) in Group 1 (n = 22) or with hCG (3000 IU, i.v.) in Group 2 (n = 20) and Group 3 (n = 22). On Day 12, the cows were treated with PG (500 micrograms in Groups 1 and 2; 1000 micrograms in Group 3). Blood samples for progesterone (P4) determination were collected on Day 12 (Experiment 1) or on Days 12 and 14 (Experiment 2). Cows were fitted with heat mount detectors and observed twice a day for signs of estrus. Four cows in Experiment 1 (1 cow each from Groups 1 and 2; 2 cows from Group 3) had plasma P4 concentrations below 1 ng/ml on Day 12 and were excluded from the analyses. In Experiment 1, cows treated with hCG or hCG + bFF had a more variable (P = 0.0007, P = 0.0005) day of occurrence of and a longer interval to estrus (5.9 +/- 0.7 d, P = 0.003 and 6.2 +/- 0.8 d, P = 0.005) than saline-treated cows (3.4 +/- 0.4 d). The plasma P4 concentrations on Day 12 were higher (P < 0.0001) in hCG- and in hCG + bFF-treated cows than in saline-treated cows (9.4 +/- 0.75 and 8.5 +/- 0.75 vs 4.1 +/- 0.27 ng/ml), but there was no correlation (P > 0.05) between plasma P4 concentrations and the interval to estrus. In Experiment 2, cows treated with hCG/500PG and hCG/1000PG had a more variable (P = 0.0007, P = 0.002) day of occurrence of and a longer interval to estrus (4.2 +/- 0.4 d, P = 0.04; 4.1 +/- 0.4 d, P = 0.03) than saline/500PG-treated cows (3.2 +/- 0.1 d). The concentrations of plasma P4 on Days 12 and 14 of both hCG/500PG- and hCG/1000PG-treated cows were higher (P < 0.05) than in saline/500PG-treated cows (7.3 +/- 0.64, 0.7 +/- 0.08 and 7.7 +/- 0.49, 0.7 +/- 0.06 vs 5.3 +/- 0.37, 0.5 +/- 0.03 ng/ml). The concentrations of plasma P4 on Days 12 or 14 and the interval to estrus were not correlated (P > 0.05) in any treatment group. The concentrations of plasma P4 on Days 12 and 14 of hCG/500PG- or hCG/1000PG-treated cows were correlated (r = 0.65, P < 0.05; r = 0.50, P < 0.05). This study indicated that treatment of cows with hCG on Day 5 of the estrous cycle reduced the synchrony of PG-induced estrus and that this reduction was not due to the failure of luteal regression.     

Waves of follicle development during the estrous cycle in sheep

The pattern of ovarian follicle development in maiden cyclic lambs was characterized using the definition of a follicle wave as the changes in the number of follicles among the days of the estrous cycle, as originally defined in cattle by Rajakoski in 1960. We also examined the steroid content relationships among follicles on Days 5 (Wave 1) and 14 (Waves 2 and 3) of the estrous cycle. In Experiment 1, the ovaries of 20 cyclic lambs (40 to 45 kg) were examined daily using transrectal ultrasonography for 1 or 2 estrous cycles (n = 31 cycles). The number of small (2 and 3 mm in diameter), medium (4 and 5 mm) and large (> or = 6 mm) follicles were aligned with the beginning and end of the average length estrous cycle and then compared among days. Identified follicles were defined as those that grew to > or = 4 mm and remained at > or = 3 mm for > or = 3 d. The number of identified follicles emerging (retrospectively identified at 2 or 3 mm) per ewe per day was also aligned with the average length estrous cycle. In Experiment 2, ewe lambs were ovariectomized on Day 5 (n = 6) or 14 (n = 5) of the estrous cycle, then follicle diameters and follicular fluid concentrations of estradiol and progesterone were compared among follicles. Data were analyzed by repeated measures ANOVA and compared among days using Fisher's LSD. In Experiment 1, either 2 (n = 10 cycles), 3 (n = 20 cycles) or 4 (n = 1 cycle) periods of emergence of identified follicles occurred during individual cycles, with estrous cycle lengths of 15.6 +/- 1.6, 16.1 +/- 1.1 and 17 d respectively. In animals with 2 or 3 periods of emergence of identified follicles, the total number of small, medium and large follicles differed (P < 0.05) among days of the estrous cycle showing a wave-like pattern. In Experiment 2, a single follicle collected on each of Days 5 and 14 of the cycle (6.2 +/- 0.2 and 3.9 +/- 0.2 mm in diameter) had a higher (P < 0.05) concentration of follicular fluid estradiol (36.2 +/- 4.4 and 50.9 +/- 21.6 ng/mL) than other follicles collected on the same day (next largest follicle: 4.3 +/- 0.3 and 3.5 +/- 0.4 mm; 4.3 +/- 0.9 and 18.2 +/- 6.7 ng/mL estradiol). The results showed that 1) there was a synchronous emergence of follicles associated with fluctuations in the number and size of follicles during the estrous cycle; 2) within a wave there was a hierarchy among follicles for diameter and steroid content; 3) ovarian follicle growth in ewe lambs occurred in 2 or 3 organized waves during the estrous cycle.     

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.     

Reduction in size of the ovulatory follicle reduces subsequent luteal size and pregnancy rate

We hypothesized that reducing the size of the ovulatory follicle using aspiration and GnRH would reduce the size of the resulting CL, reduce circulating progesterone concentrations, and alter conception rates. Lactating dairy cows (n=52) had synchronized ovulation and AI by treating with GnRH and PGF2alpha as follows: Day -9, GnRH (100 microg); Day -2, PGF2alpha (25 mg); Day 0, GnRH (100 microg); Day 1, AI. Treated cows (aspirated group; n=29) had all follicles > 4 mm in diameter aspirated on Days -5 or -6 in order to start a new follicular wave. Control cows (nonaspirated group: n=23) had no follicle aspiration. The size of follicles and CL were monitored by ultrasonography. The synchronized ovulation rate (ovulation rate to second GnRH injection: 42/52=80.8%) and double ovulation rate of synchronized cows (6/42=14.3%) did not differ (P > 0.05) between groups. Aspiration reduced the size of the ovulatory follicle (P < 0.0001; 11.5 +/- 0.2 vs 14.5 +/- 0.4 mm), and serum estradiol concentrations at second GnRH treatment (P < 0.0002; 2.5 +/- 0.4 vs 5.7 +/- 0.6 pg/mL). The volume of CL was less (P < 0.05) for aspirated than nonaspirated cows on Day 7 (2,862 +/- 228 vs 5,363 +/- 342 mm3) or Day 14 (4,652 +/- 283 vs 6,526 +/- 373 mm3). Similarly, serum progesterone concentrations were less on Day 7 (P < 0.05) and Day 14 (P < 0.10) for aspirated cows. Pregnancy rate per AI for synchronized cows was lower (P < 0.05) for aspirated (3/21=14.3%) than nonaspirated (10/21=47.6%) cows. In conclusion, ovulation of smaller follicles produced lowered fertility possibly because development of smaller CL decreased circulating progesterone concentrations.