Follicular dynamics and superovulatory response in heifers

The study examined whether the response of heifers to exogenous gonadotrophin superovulatory treatment could be predicted from a knowledge of previous antral follicular dynamics. During a pretreatment monitoring phase, of 24 normal oestrous cycles (20.1 ± 0.33 days long) observed in 17 heifers, one, 15 and seven cycles showed one, two and three antral follicular waves respectively, as measured by ultrasonography. The subsequent ovulatory response (number of corpora lutea) to ovine FSH stimulation, after a CIDR-B/oestradiol benzoate/prostaglandin analogue cycle synchronisation regime, was not correlated with either oestrous cycle length or follicle wave number during the monitoring phase or with the number of follicles observed at the start of FSH treatment, but was related to the number of follicles observed during the monitoring phase (r = 0.47, P < 0.05). In conclusion, the present results show that the outcome of FSH superovulatory stimulation in heifers cannot be predicted from a knowledge of prior follicular dynamics.     

Normal or induced secretory patterns of luteinising hormone and follicle-stimulating hormone in anoestrous gonadotrophin-releasing hormone-immunised and cyclic control heifers

The objective was to determine the effect of gonadotrophin-releasing hormone (GnRH), GnRH analogue (GnRH-A) or oestradiol administration on luteinising hormone (LH) and follicle-stimulating hormone (FSH) release in GnRH-immunised anoestrous and control cyclic heifers. Thirty-two heifers (477 ± 7.1 kg) were immunised against either human serum albumin (HSA; controls; n = 8), or a HSAGnRH conjugate. On day 70 after primary immunisation, control heifers (n = 4 per treatment; day 3 of cycle) received either (a) 2.5 μg GnRH or (b) 2.5 μg of GnRH-A (Buserelin®) and GnRH-immunised heifers (blocked by GnRH antibody titre; n = 6 per treatment) received either (c) saline, (d) 2.5 μg GnRH, (e) 25 μg GnRH or (f) 2.5 μg GnRH-A, intravenously. On day 105, 1 mg oestradiol was injected (intramuscularly) into control (n = 6) and GnRH-immunised anoestrous heifers with either low (13.4 ± 1.9% binding at 1:640; n = 6) or high GnRH antibody titres (33.4 ± 4.8% binding; n = 6). Data were analysed by ANOVA. Mean plasma LH and FSH concentrations on day 69 were higher (P < 0.05) in control than in GnRH-immunised heifers (3.1 ± 0.16 vs. 2.5 ± 0.12 ng LH ml⁻¹ and 22.5 ± 0.73 vs. 17.1 ± 0.64 ng FSH ml⁻¹, respectively). The number of LH pulses was higher (P < 0.05) in control than in GnRH-immunised heifers on day 69 (3.4 ± 0.45 and 1.0 ± 0.26 pulses per 6 h, respectively). On day 70, 2.5 μg GnRH increased (P < 0.05) LH concentrations in control but not in GnRH-immunised heifers, while both 25 μg GnRH and 2.5 μg GnRH-A increased (P < 0.05) LH concentrations in GnRH-immunised heifers, and 2.5 μg GnRH-A increased LH in controls. FSH was increased (P < 0.05) in GnRH-immunised heifers following 25 μg GnRH and 2.5 μg GnRH-A. Oestradiol challenge increased (P < 0.05) LH concentrations during the 13–24 h period after challenge with a greater (P < 0.05) increase in control than in GnRH-immunised heifers. FSH concentrations were decreased (P < 0.05) for at least 30 h after oestradiol challenge. In conclusion, GnRH immunisation decreased LH pulsatility and mean LH and FSH concentrations. GnRH antibodies neutralised low doses of GnRH (2.5 μg), but not high doses of GnRH (25 μg) and GnRH-A (2.5 μg). GnRH immunisation decreased the rise in LH concentrations following oestradiol challenge.     

Follicular development and reproductive endocrinology during and after superovulation in heifers and mature cows displaying contrasting superovulatory responses

To understand the causes for poor response to superovulation in mature cows of high genetic potential, endocrine and follicular events during and after superovulation were compared in heifers (<2 yr old) yielding large numbers of embryos and cows (9 to 13 yr old) known to be poor embryo donors. Follicular development was monitored by daily ultrasonography. Blood samples were taken 2 to 3 times a day for the measurements of P4, E2, FSH and LH by RIA. Intensive blood collections at 15-min intervals for 6 h were also performed during preovulatory and luteal phases. The number of embryos produced in the heifers (15.2 +/- 2; mean +/- SEM) and the cows (0.6 +/- 0.4), was similar to the number of ovulatory follicles derived from ultrasonographic observations in the heifers (16.2 +/- 3.7), but not in the cows (7.8 +/- 2.8). Contrary to that observations in heifers, there was no increase in the number of 4- to 5-mm follicles in cows during superovulation. The number of larger follicles (>5 mm) increased during superovulation in both cattle groups, but it was significantly lower in cows than in heifers. During superovulation, the maximal E2 concentration was greater (P < 0.0001) in heifers than in cows. One cow showed delayed luteolysis during superovulation, while another had abnormally high FSH (>10 ng/ml) and LH (>3 ng/ml) concentrations following superovulation. All the cows had a postovulatory FSH rise which was not detected in the heifers. The results showed that attempts to improve superovulatory response in mature genetically valuable cows are hampered by a number of reproductive disorders that are not predictable from the study of the unstimulated cycle.     

Role of increased estradiol on altering the follicle diameters and gonadotropin concentrations that have been reported for double-ovulating heifers

During the preovulatory period in heifers that ovulate from two compared to one follicle, circulating concentrations of estradiol-17β (E2) are greater, diameter of follicles and concentration of FSH are reduced, and the LH surge occurs sooner. The effect of increased E2 on the reported characteristics of double ovulation was studied by treating heifers with 0.07 mg E2, 0.09 mg E2, or vehicle in four treatments at 6-h intervals (n=6 heifers/group), beginning at the time of expected follicle deviation (largest follicle, 8.5mm). There were no significant differences on follicle diameters or hormone concentrations between the 0.07 and 0.09 mg E2 groups, and heifers were combined into one E2 group (n=12). The E2 treatments induced concomitant preovulatory surges in LH and FSH at 34.0 ± 2.6h after first treatment, compared to 57.6 ± 4.5h in the vehicle group (P<0.0002). The E2 treatments did not affect FSH concentrations during the preovulatory gonadotropin surge. The diameter of the preovulatory follicle at the LH peak was smaller (P<0.0001) in the E2-treated group (10.2 ± 0.2mm) than in the vehicle group (13.1 ± 0.6mm). The hypothesis was not supported that the previously reported increase in circulating E2 in heifers with double preovulatory follicles accounts for the reported lesser concentrations in the preovulatory FSH surge in heifers with double ovulations. Hypotheses were supported that the reported earlier occurrence of the preovulatory LH surge and smaller preovulatory follicles in heifers with double ovulations are attributable to the reported increase in E2 from the double preovulatory follicles.     

Effects of active immunization of prepubertal heifers against prostaglandin F2α, on the onset of puberty and subsequent ovarian function

The objective of these two experiments was to determine the effect of immunization of prepubertal heifers against prostaglandin F_2α conjugated to human serum albumin (PGF-HSA) on the onset of puberty and subsequent ovarian function. Heifers that were 2–3 months of age (n=34; mean ± standard error of the mean (SEM) bodyweight 75 ± 1.4 kg) were given a primary immunization (Day 0) and a booster 183 days later using one of two adjuvants; non-ulcerative Freund's adjuvant (NUFA) and diethylaminoethyl-dextran (DEAF-dextran) and one of two doses of PGF-HSA as immunogen (1 and 10 mg), with six to seven heifers per treatment; control heifers (n=7) were untreated. The experiment lasted up to 562 days. Plasma antibody titres every 2–3 weeks and progesterone concentrations two to three times weekly were determined. Oestrous behaviour was observed twice daily. In addition, the follicular status of ovaries of cyclic (n=4), prepubertal (n=6) or prolonged luteal phase ( > 200 days, induced by immunization against PGF) heifers (n=5) were monitored daily for 23 consecutive days by ultrasound examination. Antibody titres were higher (P < 0.05) immediately following booster immunization in heifers immunized with DEAF-dextran than with NUFA. High titres were maintained for longer (P < 0.05) in heifers immunized with NUFA than with DEAF-dextran. The mean number of oestrous periods was decreased (P < 0.05) in PGF-immunized heifers in comparison with control heifers following booster immunization (3.1, 0.5, 0.9, 1.5, 1.1 in control heifers and in heifers immunized with NUFA-1 mg, NUFA-10 mg, DEAE-dextran-1 mg and DEAE-dextran-10 mg, respectively. Puberty was delayed (P < 0.05), relative to control heifers, by 185 and 122 days (pooled standard error of the difference, 89) in heifers immunized with NUFA-1 mg and DEAE-dextran-10 mg, respectively. After first ovulation at puberty, persistent corpora lutea (CL) formed in two of six heifers immunized with NUFA-1 mg, five of seven immunized with NUFA-10 mg, two of seven immunized with DEAE-dextran-1 mg and one of seven immunized with DEAE-dextran-10 mg. The duration of persistence was 103 ± 19, 301 ± 33, 124 ± 23 and 100 days, respectively. The mean (±SEM) number of dominant follicles was not different (P > 0.05) for the three groups of heifers that were examined by ultrasound (3.7 ± 0.2, 3.8 ± 0.3 and 3.2 ± 0.3 for cyclic, prepubertal and persistent CL, respectively); neither was their rate of growth (1.8 ± 0.2, 1.5 ± 0.1 and 1.5±0.2 mm day⁻¹, respectively), rate of atresia (1.3 ± 0.1, 1.6 ± 0.1 and 1.8 ± 0.2 mm day⁻¹, respectively) or the maximum size of dominant follicles (11.2 ± 0.4, 9.6 ± 0.3 and 12.4 ± 1.8 mm, respectively). In summary, immunization of prepubertal heifers against PGF delayed the onset of puberty and reduced subsequent oestrous behaviour due to the formation of persistent CL following first ovulation and there was a similar growth pattern of dominant follicles before first ovulation in prepubertal heifers, cyclic heifers and heifers which have long-term persistent CL.     

Follicular populations during the estrous cycle in heifers. II. Influence of right and left sides and intraovarian effect of the corpus luteum

Ovarian follicles ≥2 mm were studied in 22 Holstein heifers by daily ultrasound examinations. Data were partitioned by right vs. left ovary and corpus luteum bearing ovary vs. the contralateral ovary. There were significantly more (P < 0.03) follicles 4–6 mm, > 13mm and total ≥2 mm in the right ovary, regardless of the presence of a corpus luteum. Significantly more (P < 0.05) follicles 2–3 mm, > 13 mm and total ≥2 mm were observed in the ovary bearing the corpus luteum. Interactions between day and corpus luteum appeared to be due to a greater number of follicles in the ovary bearing the corpus luteum during the first part of the interovulatory interval. There was also a day by right side vs. left side interaction for the number of follicles > 13 mm. Interpretation of the interactions was that the presence of a corpus luteum was conducive to the development of more anovulatory diestrous follicles > 12 mm. However, as regression of the corpus luteum progressed, there was an apparent proclivity for preovulatory follicular development in the right ovary. There was no apparent pattern of alternating sides of ovulation or of alternating sides of development of anovulatory diestrous follicles and preovulatory follicles in heifers observed for more than one interovulatory interval. There was not a significant difference in the maximum diameter attained by the anovulatory diestrous follicle or preovulatory follicle between ovaries ipsilateral or contralateral to the corpus luteum; however, the maximum diameter attained by the preovulatory follicle was greater (P < 0.05) than that attained by the anovulatory diestrous follicle.     

Oxytocin,prostaglandins E and F,estradiol, progesterone,sodium, and potassium in preovulatory bovine follicles either developed normally or stimulated by follicle stimulating hormone

To determine the differences between stimulation by follicle stimulating hormone (FSH) and normal development of follicles in heifers and the endocrinologic events associated with ovulatory development of follicles in heifers, follicular fluid was aspirated from follicles (n = 627) at 24, 48, and 72 h after estrus synchronization (via prostaglandin F_2α (PGF_2α)) from control animals (n = 10/time period) and a treatment group (n = 10/time period) that received FSH. No endocrinologic or ionic differences were noted between follicles harvested from control or FSH-stimulated animals within follicular size or time (24, 48, or 72 h). No interaction of treatment (FSH stimulation vs controls) by time was detected; thus the effect and timing of the LH surge was similar between treatments.At 48 h after PGF_2α administration, sodium from follicular fluid decreased and potassium increased, signifying a considerable physiologic change in the follicle. Follicular prostaglandins E and F increased ten-fold by 72 h, and these changes were primarily found in the estrogen-inactive follicles at 72 h. Though progesterone concentrations within follicular fluid tended to increase throughout the three periods and increased with enlargement of follicular size, a major increase was detected 72 h after PGF_2α injection. Estradiol concentrations tended to increase with enlargement of follicular size within a time period, but estradiol concentrations in the follicles greater than 10 mm in diameter decreased with time (24, 48, and 72 h) after PGF_2α. Concentrations of oxytocin increased with increases in follicular diameter, and the time trends were similar to changes in follicular progesterone. By 72 h after PGF_2α administration, follicular estradiol) was decreasing in concentration and progesterone and oxytocin were increasing, thus signifying a change in the secretory role of the granulosa cells.     

Alterations in intrafollicular regulatory factors and apoptosis during selection of follicles in the first follicular wave of the bovine estrous cycle

Changes in follicular fluid (FF) concentrations of estradiol, inhibin forms, and insulin-like growth factor binding proteins (IGFBPs), percentage of apoptotic granulosa cells (%A), and follicular size for individual follicles in a growing cohort were determined throughout the first wave of follicular development during the bovine estrous cycle and related to FSH decline. Four groups of heifers (n = 31) were ovariectomized between Days 1.5 and 4.5 of the estrous cycle at 5 +/- 1, 33 +/- 2, 53 +/- 1, and 84 +/- 2 h after the periovulatory peak in FSH concentrations. Follicles > or = 2.5 mm were dissected, measured, and FF aspirated. The five largest follicles were ranked based on their diameter (F1 to F5). Diameters of F1 to F5 were positively correlated with interval from FSH peak (r > or = 0.6, P < 0.05). Five hours after the FSH peak, follicular diameter and FF concentrations of estradiol, inhibins, and IGFBPs were similar for F1 to F5. From 5 to 33 h, amounts of the six precursor inhibin forms (> or = 48 kDa) increased (P < 0.05) in F1 follicles. The IGFBPs in F1 follicles remained low at all time periods. At 33 h, amounts of IGFBP-4 and -5 were higher (P < 0.05) in F4 and F5 compared with F1 follicles. At 84 h, IGFBP-2, -4, and -5 were increased (P < 0.05) in F3, F4, and F5 compared with F1. At 5, 33, or 53 h, %A was not different between follicles in any size class. At 84 h %A was increased (P < 0.05) in follicles <6 mm in diameter. However, at that time, %A did not differ between the selected DF and the largest subordinate follicle. For individual heifers, the selected DF at 84 h was largest in size, highest in estradiol, and lowest in IGFBP-2 and -4. The F1 follicle had highest estradiol in 23 of 27 heifers irrespective of stage of the wave and lowest IGFBP-4 in 19 of 21 heifers from 33 h. We concluded that the earliest intrafollicular changes that differentiate a dominant-like follicle from the growing cohort are enhanced capacity to produce estradiol and maintenance of low levels of IGFBPs.     

Long-term in vitro culture of ovarian cortical tissue in goats: effects of FSH and IGF-I on preantral follicular development and FSH and IGF-I receptor mRNA expression

Long-term in vitro culture (16?days) of caprine ovarian cortical tissue was performed to test the effect of FSH and IGF-I on the viability and development of preantral follicles and mRNA expression for FSH and IGF-I receptors. Fragments were cultured in ??-MEM⁺ alone or supplemented with different combinations of FSH and IGF-I (sequential medium). The culture period was divided into two parts. Follicles were isolated and classified as normal or abnormal and primordial, primary or secondary. Viability of isolated follicles was determined by staining with Trypan Blue dye. Expression of FSHR and IGFR-1 mRNA was evaluated by qPCR. At day 8 of culture, more (P?<?0.05) follicles in treatments containing IGF-I alone or associated with FSH were normal and viable (overall mean, 81?% and 79?% respectively) than the treatments cultured with FSH or ??-MEM⁺ alone (68?% and 63?%). At day 16 of culture, treatments with FSH and/or IGF-I had more (P?<?0.05) viable follicles (69?%) than ??-MEM⁺ (38?%). The percentages of follicular development observed in the IGF-I/FSH, FSH+IGF-I/FSH+IGF-I and FSH/IGF-I treatments were similar but higher (P?<?0.05) than the other treatments. FSH and IGF-I during the entire culture period maximized (P?<?0.05) follicular and oocyte diameters and the percentage of secondary follicles (28?%). FSHR mRNA expression in the non-cultured control was similar to the treatment supplemented with FSH and IGF-I but higher (P?<?0.05) than ??-MEM⁺. IGFR-1 expression did not differ among treatments. Association of FSH and IGF-I in long-term in vitro culture promoted follicular development, maintaining FSHR mRNA expression.     

Follicular dynamics prior to and during superovulation in heifers

The present ultrasonographic study examined the relationship between certain follicular parameters and the superovulatory response in gonadotropin-stimulated heifers. Thirty heifers received a total of 35 mg FSH twice daily for 4 d and 0.75 mg cloprostenol were given to induce luteolysis and estrus at 72 h after the initial FSH injection. Transrectal ultrasonography was performed once daily from 1 or 2 d before the initial FSH injection and until the day of estrus. The number of small (2 to 4 mm), medium (5 to 9 mm), and large (>/=10 mm) size follicles as well as the diameter of the large follicles were recorded. Embryos were recovered non-surgically 6 or 7 d after estrus, and the number of corpora lutea was determined by palpation per rectum. Heifers with >2 or 0.05). The number of large follicles and the sum of medium and large follicles were positively correlated (r=0.43 and r=0.54, respectively; P<0.05) with the number of corpora lutea palpated on the day of embryo recovery (6 to 7 d after estrus). In conclusion, there was an effect of the day relative to initiation of FSH treatment on all follicular categories in heifers responding positively to superovulation, and there was no effect of side (left or right ovary) or of corpus luteum diameter (ipsilateral or contralateral).     

Concentrations of inhibins and steroids in follicular fluid during development of dominant follicules in heifers

The objective of this study was to examine changes in intrafollicular concentrations of inhibins and steroids in heifers during growth of dominant follicles. To obtain dominant ovulatory follicles, heifers received injections of prostaglandin (PG) on Day 9 of an estrous cycle and were ovariectomized (OVX) 0, 24, 48, 60, or 72 h after injection. To obtain dominant nonovulatory follicles, heifers were OVX on Day 3, 6, or 9 of a cycle. Follicular size was determined, follicular fluid (FF) was collected from follicles 6 mm or greater in diameter, and RIA was used to quantify concentrations of inhibins, estradiol, and progesterone in FF. During growth of dominant ovulatory follicles, concentrations of estradiol and progesterone increased, whereas inhibins decreased when compared with dominant follicles on Day 9 before PG treatment. Concentrations of inhibins were inversely correlated with size and concentrations of estradiol in dominant ovulatory follicles. As dominant nonovulatory follicles increased in size, concentrations of inhibins, estradiol, and progesterone increased. Concentrations of inhibins were positively correlated with size and with progesterone concentrations in dominant nonovulatory follicles. Concentrations of inhibins were greater in dominant nonovulatory follicles than in atretic follicles. In summary, intrafollicular concentrations of inhibins decreased during growth of dominant ovulatory follicles, but increased during growth of dominant nonovulatory follicles. Because of the well-known suppressive action of inhibins on FSH secretion, we hypothesize that inhibins are involved in growth and atresia of dominant follicles during the bovine estrous cycle.     

Two-way coupling between FSH and the dominant follicle in heifers

Follicular Wave 1 and 2 and the associated FSH Surge 1 and 2 were used to designate the first two waves and surges of the interovulatory interval in two experiments in heifers. In experiment 1, a group with early (group E, N = 9) and late (group L, N = 5) development of the dominant follicle of Wave 1 were used as natural models to study FSH/follicle coupling. The day of wave emergence and the day of deviation in diameters between the two largest follicles were not different between groups. Emergence of Wave 2 and maximal FSH concentration in Surge 2 was approximately 1 day later (P < 0.03) in group L. Diameter of the dominant follicle of wave 1 (13.8 ± 0.3 mm vs. 12.0 ± 0.3 mm) and FSH concentrations in Surge 2 (0.29 ± 0.02 ng/mL vs. 0.21 ± 0.03 ng/mL) were first greater (P < 0.05) in group E than in group L at 4 and 5 days, respectively, after wave emergence. In experiment 2, treatment with estradiol (N = 8) when the dominant follicle of Wave 1 was ≥11 mm (Hour 0) resulted in a decrease (P < 0.02) in FSH and slower (P < 0.05) growth rate of the follicle between Hours 0 and 4. Results supported the following hypotheses: (1) the FSH surge that stimulates emergence of a follicular wave is associated with final growth of the dominant follicle of the previous anovulatory wave; and (2) suppression of FSH Surge 2 when the dominant follicle of Wave 1 is ≥11 mm is associated with a decrease in diameter. It is concluded for the first time that two-way FSH/follicle coupling in heifers continues during final growth of the dominant follicle of Wave 1 and that Surge 2 is the FSH source.     

Effect of inhibin immunisation using different synthetic peptide fragments of the bovine αc-subunit on plasma anti-inhibin titres,plasma FSH concentrations and the incidence of multiple ovulation in heifers

In an effort to improve the effectiveness of inhibin immunisation in promoting multiple ovulation in cattle and to clarify the mechanism(s) involved, heifers (n = 5 per group) were immunised against ovalbumin conjugates of different synthetic peptide sequences of the α_c-subunit of bovine inhibin (bIα) selected using antigenic prediction methods. Plasma inhibin antibody titre (percentage binding of ¹²⁵I-labelled M_r 32 000 native bovine inhibin), plasma follicle-stimulating hormone (FSH) concentration and ovulatory response (number of corpora lutea observed by transrectal ovarian ultrasonography) were recorded over a 16 week period.Heifers immunised against the bIα1–29 and 63–72 peptides (alone or in combination), had relatively high anti-inhibin titres (over 7.5% binding) and showed a significantly (P < 0.05) increased incidence of multiple ovulations (18–65%) compared with ovalbumin-immunised controls. However, immunisation against the 1–16 and 108–123 peptides was relatively ineffective in generating antibodies reactive with native inhibin (less than 7.5% binding) and gave little or no increase in incidence of multiple ovulations (0–10%).Analysis of results for all 33 heifers revealed a significant linear relationship between mean inhibin antibody titre and mean plasma FSH concentration (r = 0.42; P < 0.02) and between mean inhibin antibody titre and incidence of multiple ovulation (r = 0.89; P < 0.0001). A significant quadratic relationship existed between mean inhibin antibody titre and the mean number of ovulations per cycle (r = 0.88; P < 0.0001). However, partial correlation analysis showed a highly significant association between anti-inhibin titre and ovulatory response which was independent of changes in mean plasma FSH concentrations.These results extend previous studies involving inhibin peptide-immunised cattle by showing that the magnitude of the ovulatory response is directly related to the prevailing titre of antibodies reactive with native inhibin. However, they do not support the hypothesis that the ovulatory response is mediated solely by a rise in FSH secretion.     

Differential abundance of IGF1, bile acids,and the genes involved in their signaling in the dominant follicle microenvironment of lactating cows and nulliparous heifers

It is well documented that incidence of fertility problems is high in lactating cows but not in heifers of the same genetic merit. Understanding the metabolic and molecular differences between fertile heifers and relatively infertile lactating cows will help us understand the pathogenesis of infertility in dairy cows. Follicular waves in lactating cows (30–50 days in milk; n = 12) and heifers (n = 10) were synchronized by ultrasound-guided follicle ablation. Follicular fluid and granulosa cells of the dominant follicle were collected by ultrasound-guided aspiration along with blood sampling on Day 6 after synchronization. Dominant and subordinate follicles were larger in lactating cows than in heifers. Metabolic stress in lactating cows was evidenced by lower glucose and higher ß-hydroxy butyric acid compared with heifers. Insulin-like growth factor 1 signaling was reduced in the dominant follicle in lactating cows through reduced insulin-like growth factor 1 concentrations in plasma and follicular fluid of the dominant follicle, and reduced expression of pregnancy-associated plasma protein A (PAPPA) in their granulosa cells. We also found increased levels of total bile acids in the follicular fluid of the dominant follicle of lactating cows compared with heifers. Granulosa cells of the dominant follicle had higher expression of SLC10A2 and GPBAR1 (bile acid transporter and receptor, respectively) in lactating cows. These novel data are indicative of increased bile acid signaling within the dominant follicles of lactating cows compared with heifers. Overall, we demonstrate in the present study the metabolic, endocrine, and molecular differences within the microenvironment of the dominant follicles in lactating cows and heifers. These differences in follicular microenvironment may contribute toward abnormal ovarian function in lactating dairy cows.     

Supplementation of 17β-estradiol and progesterone in the co-culture medium of bovine oviductal epithelial cells and ovine spermatozoa reduces the sperm kinematics and capacitation

This study investigated the effect that bovine oviductal epithelial cell (BOEC) and ovine spermatozoa co-culture exposed to different hormonal environments had on ram sperm function over the course of a 24-h incubation period. Ram cooled-stored spermatozoa were selected by swim-up and then co-cultured separately for 24?h at 38.5?°C under 5% CO₂ with either: (1) Fert-TALP medium (positive control [POSControl]), (2) Fert-TALP medium supplemented with 17β-estradiol (E2) and progesterone (P4) at concentrations similar to follicular phase (Follicular NEGControl), (3) Fert-TALP medium supplemented with E2 and P4 concentrations similar to luteal phase (Luteal NEGControl), (4) BOEC cultured in the same medium as that of the Follicular NEGControl group (Follicular BOEC group), or (5) BOEC cultured in the same medium as that of the Luteal NEGControl group (Luteal BOEC group). The sperm kinematics, capacitation status, and plasma membrane (PM) integrity were evaluated at different intervals. Sperm PM integrity was not affected (P ? 0.05) by BOEC co-culture, regardless of the phase of the estrous cycle. After 4?h of incubation, the Luteal BOEC group presented lower (P?<? 0.05) progressive motility and total motility than the Luteal NEGControl group while the Follicular BOEC group showed lower (P?<? 0.05) velocimetric parameters and progressive motility than the Follicular NEGControl group. Throughout the incubation period, both BOEC co-culture groups showed a decrease (P?<? 0.05) in their capacitation rate in comparison to the POSControl group. Conversely, the Luteal BOEC group presented a higher (P?<? 0.05) non-capacitated rate than both the POSControl and Luteal NEGControl groups. In conclusion, BOEC co-culture with ovine spermatozoa at either the follicular or luteal phase decreases sperm kinematics and delays sperm capacitation.     

Regulation of the proliferative activity of ovarian surface epithelial cells by follicular fluid

Despite critical roles of the ovarian surface epithelium (OSE) in ovulation and post-ovulatory wound repair, little is known about the physiological mechanism regulating OSE proliferation. A role of follicles and corpora lutea in locally regulating the proliferative activity of OSE has been suggested. In this study, the effects of follicular and luteal products on proliferation of cultured OSE cells were tested using cells obtained from seasonally anoestrous ewes. Follicular fluid but not luteal extracts induced OSE cell proliferation (2.5-fold relative to untreated controls; P < 0.0001). The response of OSE cells was not affected by follicle size or previous charcoal-extraction of follicular fluid (P > 0.1). Treatment with IGF-1 (2.2-fold; P < 0.01), EGF (1.9-fold; P < 0.01) and, to a lesser extent, FSH (P < 0.05) also induced OSE cell proliferation. In contrast, oestradiol or progesterone did not induce cell proliferation or enhance the effects of FSH on proliferation (P > 0.1). It was concluded that follicular fluid can directly stimulate ovine OSE cell proliferation and that this effect is attributable to non-steroidal mitogens.     

Influence of age,body weight and body condition score before mating start date on the pubertal rate of maiden Holstein–Friesian heifers and implications for subsequent cow performance and profitability

The aim of this study was to assess the influence of age, body weight (BW) and body condition score (BCS) of maiden Holstein–Friesian heifers before mating start date (MSD) on the rate of puberty, subsequent production and longevity and their implications with regard to farm profitability. Data were available on 871 Holstein–Friesian heifers from 48 herds. BW was recorded electronically and BCS was recorded by a single operator on a scale of 1 to 5. Heifer age was calculated as the number of days from birth to the day of visit. All of the independent variables of interest were grouped into three or four categories. Three age categories (thirtiles), four BW categories (quartiles) and four BCS categories (⩽2.75, 3.00, 3.25 and ⩾3.50) were formed. Heifers with an identifiable corpus lutuem (CL) in the presence or absence of large follicles and peri-ovulatory signs and with a plasma progesterone (P4) concentration ⩾1 ng/ml were classified as pubertal. In addition, heifers without an identifiable CL in the presence or absence of large follicles and peri-ovulatory signs but with a P4 concentration ⩾1 ng/ml were also classified as pubertal. Age, BW and BCS at MSD were all found to be significantly associated with pubertal rate (P < 0.05). Age was shown to have no practical implications on subsequent cow performance. BW at MSD was favourably associated with subsequent calving date (P < 0.05), subsequent cow BW (P < 0.001) and potential (305 days) milk fat plus protein yield (P < 0.001). BCS at MSD was found to be favourably associated with milk fat plus protein yield potential (P < 0.05) and BCS (P < 0.001) during lactation. The economic analysis undertaken indicated that larger, well-grown heifers will be more profitable because of superior production potential, all else being equal. However, because of the finding of poorer reproductive efficiency in heifers grown to more than 343 kg at MSD, heifers at ∼330 kg at MSD are deemed optimal. This will correspond to mature cow BW of ∼550 kg.     

Impacts of stocking density on development and puberty attainment of replacement beef heifers

In all, 60 Angus×Hereford heifers were ranked by age and BW (210±2 days and 220±2 kg) on day 0, and assigned to: (a) one of three drylot pens (10×14 m pens; 10 heifers/pen) resulting in a stocking density of 14 m²/heifer (HIDENS; n=3), or (b) one of three pastures (25 ha pastures; 10 heifers/pasture), resulting in a stocking density of 25 000 m²/heifer (LOWDENS; n=3). Pastures were harvested for hay before the beginning of this experiment, and negligible forage was available for grazing to LOWDENS heifers during the experiment (days 0 to 182). All heifers received the same limited-fed diet, which averaged (dry matter basis) 4.0 kg/heifer daily of hay and 3.0 kg/heifer daily of a corn-based concentrate. Heifer shrunk BW was recorded after 16 h of feed and water withdrawal on days −3 and 183 for BW gain calculation. On day 0, heifers were fitted with a pedometer behind their right shoulder. Each week, pedometer results were recorded and blood samples were collected for puberty evaluation via plasma progesterone. Plasma samples collected on days 0, 28, 56, 84, 112, 140, 161 and 182 were also analyzed for cortisol concentrations. On days 0, 49, 98, 147 and 182, hair samples were collected from the tail switch for analysis of hair cortisol concentrations. On days 28, 102 and 175, blood samples were collected for whole blood RNA isolation and analysis of heat shock protein (HSP) 70 and HSP72 mRNA expression. Heifers from LOWDENS had more (P<0.01) steps/week compared with HIDENS. No treatment effects were detected (P=0.82) for heifer BW gain. Plasma cortisol concentrations were greater (P⩽0.05) in LOWDENS compared with HIDENS heifers on days 84, 140, 161 and 182 (treatment×day interaction; P<0.01). Hair cortisol concentrations were greater (P<0.01) in HIDENS compared with LOWDENS heifers beginning on day 98 (treatment×day interaction; P<0.01). Heifers from LOWDENS had greater (P=0.04) mean mRNA expression of HSP72, and tended (P=0.09) to have greater mean mRNA expression of HSP70 compared with HIDENS. Heifers from HIDENS experienced delayed puberty attainment and had less (P<0.01) proportion of pubertal heifers on day 182 compared with LOWDENS (treatment×day interaction; P<0.01). In summary, HIDENS altered heifer stress-related and physiological responses, and delayed puberty attainment compared with LOWDENS.     

Oocyte structure and follicular steroid concentrations in superovulated versus unstimulated heifers

A highly variable yield of viable embryos in superovulated cattle is a major hindrance to the embryo transfer industry. To trace the cause of this problem, investigations were carried out on the intrafollicular steroids and structure of oocytes originating from follicles of follicular stimulating hormone (FSH)-stimulated (superovulated) and unstimulated heifers. Unstimulated heifers were slaughtered at midcycle, or administered cloprostenol (PG) at midcycle and slaughtered after 24, 48, or 72 hr, while superovulated heifers were administered 4 injections of pFSH (2 injections per day) and slaughtered 12 hr later, or administered 6, 7, or 8 injections of FSH in combination with PG at the 5th and 6th injection, and slaughtered 24, 36, or 60 hr, respectively, after the first PG injection. The follicular fluid from the largest (presumptive dominant) follicle of the unstimulated heifers and from potentially ovulatory follicles (≥8 mm in diameter) of the superovulated heifers were assayed for estradiol-17β (E2) and progesterone (P4), while the oocyte cumulus complexes from such follicles were processed for transmission electron microscopy. The mean E2 and especially P4 concentrations of the potentially ovulatory follicles of the superovulated heifers were lower than similar follicles of the unstimulated animals (83.7 ± 76.7 ng/ml vs. 208.1 ± 357.0 ng/ml, P > 0.05 and 31.1 ± 38.7 ng/ml vs. 150.3 ± 202, P < 0.05, respectively). The unstimulated oocytes had, in general, spherical oocyte nuclei and compact nucleoli before PG administration, while after PG, undulation of the nuclear envelope and nucleolus vacuolization was characteristic. The superovulated oocytes, in comparison, displayed the following deviations: premature perivitelline space formation, lack of nucleolar vacuolization, reduced amount of lipid droplets and lack of lipid-mitochondria association, enlarged rough endoplasmic reticulum compartment, and increased condensation of chromatin and elongation, i.e., expansion of some cumulus cells. Degenerative oocytes were only found in the superovulated group. It is concluded that FSH-stimulation is associated with reduced intrafollicular E2 and P4 concentrations and subcellular deviations in the oocytes that are established early in the superovulatory process. These deviations may contribute to the reduced developmental competence of superovulated oocytes. © 1994 Wiley-Liss, Inc.     

Roles of kisspeptin in IVF/ICSI-treated infertile women and in human granulosa cells

Kisspeptin, a crucial central regulator of reproduction, has been used as a trigger in in vitro fertilization (IVF) treatment. This study aimed to investigate the roles of kisspeptin in IVF treatment in infertile females (n = 30); and in steroidogenesis in human granulosa-like tumor cell line (KGN). In the human study, blood was collected at three time points including (1) the beginning of gonadotropin stimulation (Phase I), (2) around eight days after gonadotropin stimulation (Phase II), and (3) on the day of ovum pick-up (Phase III). Follicular fluid (FF) was collected at Phase III. Serum human chorionic gonadotropin (hCG) was measured 15 days after embryo transfer and fetal heart beats were determined around 42 days of menstrual cycle to classify the subjects into successful and unsuccessful groups. FF kisspeptin levels were higher in successful compared with unsuccessful subjects (P < 0.01). Kisspeptin levels were significantly higher in FF than in serum in successful subjects (P < 0.05) but were comparable in unsuccessful subjects. Serum kisspeptin was comparable among three phases in the successful group but its levels in Phase III were significantly lower compared with Phase I in the unsuccessful group (P < 0.01). Serum kisspeptin in Phase II/III had positive correlations with serum E2 in Phases II and III and the outcomes of IVF/intracytoplasmic sperm injection (ICSI) treatment including serum hCG levels. For the cell experiment (n = 3), kisspeptin treatment in the presence of FSH together with IGF-1 enhanced CYP19A1 (aromatase) mRNA expression compared with control. FSH alone increased aromatase concentrations in the supernatant compared with control and kisspeptin at the dose of 10^-2 mmol/L with FSH enhanced aromatase concentrations in the supernatant compared with FSH alone (P < 0.001 all). In conclusion, kisspeptin enhanced aromatase expression and secretion and was associated with positive outcomes of IVF/ICSI treatment. Further studies regarding supplementation of kisspeptin could reveal its beneficial effects on IVF/ICSI treatment.