The aim of this study was to characterize the immediate effects of heat stress on plasma FSH and inhibin concentrations, and its involvement in follicular dynamics during a complete oestrous cycle, and to examine a possible delayed effect of heat stress on follicular development. Holstein dairy cows were oestrous synchronized and randomly assigned to either cooled (n = 7) or heat-stressed (n = 6) treatment groups. During a complete oestrous cycle, control cows, which were cooled, maintained normothermia, whereas heat-stressed cows, which were exposed to direct solar radiation, developed hyperthermia. At the end of this oestrous cycle (treated cycle), both groups were cooled and maintained normothermia for the first 10 days of the subsequent oestrous cycle. Throughout this period, follicular development was examined by ultrasonography, and plasma samples were collected. During the second follicular wave of the treated oestrous cycle, a significantly larger cohort of medium sized follicles (6-9 mm) was found in heat-stressed cows than in cooled cows (P < 0.05). The enhanced growth of follicles in this wave in heat-stressed cows was associated with a higher plasma FSH increase which lasted 4 more days (days 8-13 of the oestrous cycle; P < 0.05), and coincided with a decrease in the plasma concentration of immunoreactive inhibin (days 5-18 of the oestrous cycle; P < 0.05). During the follicular phase (days 17-20 of the treated cycle), heat-stressed cows showed an increase in the number of large follicles (>/= 10 mm), and the preovulatory plasma FSH surge was significantly higher in heat-stressed cows than in cooled cows (P < 0.01). The effect of heat stress was also observed during the first follicular wave of the subsequent cycle: the postovulatory plasma FSH concentration was higher (P < 0.01), but fewer medium follicles developed, and the first follicular wave decreased at a slower rate in previously heat-stressed cows than in cooled cows (0.40 and 0.71 follicles per day, respectively). This study shows both immediate and delayed effects of heat stress on follicular dynamics, which were associated with high FSH and low inhibin concentrations in plasma. These alterations may have physiological significance that could be associated with low fertility of cattle during the summer and autumn.
Z Roth, R Meidan, R Braw-Tal and D Wolfenson
E. Gilad, R. Meidan, A. Berman, Y. Graber and D. Wolfenson
Effects of acute and seasonal heat stress on tonic and GnRH-induced LH and FSH secretion were examined during the early follicular phase of the oestrous cycle of cows (n = 40). Prostaglandin F2α was injected on day 11 ± 1 of the oestrous cycle and on the next day blood samples were collected at intervals of 15–20 min for 14 h, and i.m. injection of GnRH was given after 7 h. Treatments compared were control versus acute heat stress during blood sampling in winter, and cooled versus chronic heat stress in summer. Before GnRH injection, chronic heat stress in summer did not affect basal concentrations of plasma LH, but did lower LH pulse amplitude. However, in cows with low plasma oestradiol (1.9 ± 0.2 pg ml−1), the mean and basal concentrations and amplitude of tonic LH pulses were reduced by heat stress (3.1, 2.1 and 4.8 versus 1.9, 1.4 and 2.5 ng ml−1, respectively). In cows with high plasma oestradiol (6.3 ± 0.5 pg ml−1), these parameters were not affected. In chronically heat stressed cows in summer, GnRH-induced increases in plasma LH and FSH concentrations were the same as in the cooled controls. However, in cows with low plasma oestradiol, mean concentrations of FSH in plasma (31.8 versus 25.5 ng ml−1), the peak of the GnRH-induced FSH and LH surge (FSH 47.4 versus 35.6 ng ml−1, LH 50.7 versus 37.3 ng ml−1) and the shape of the GnRH-induced FSH and LH curves (treatment by time interaction) were significantly lower in non-cooled versus cooled controls. The GnRH-induced increase in LH secretion was unaffected by chronic heat stress in cows with high concentrations of oestradiol in plasma. In winter, acute heat stress depressed the mean concentration of FSH in plasma and decreased the GnRH-induced release of FSH in cases with low but not with high concentrations of oestradiol in plasma. The peak of the GnRH-induced surge of LH in all acutely heat stressed cows was significantly lower in winter than in control cows, irrespective of concentrations of oestradiol in plasma. These results show that heat stress affects the secretion of gonadotrophins more in cows with low concentrations of oestradiol than in those with high concentrations of oestradiol in plasma.
D. Wolfenson, W. W. Thatcher, M. Drost, D. Caton, D. B. Foster and M. M. LeBlanc
Summary. Holstein or crossbred beef cows were anaesthetized on Days 15 to 17 after oestrus; the ovarian artery (OA), ovarian (utero-ovarian) vein (OV) and a peripheral artery (PA), were catheterized for chronic blood sampling. Beginning on the day after surgery, 6 sequential blood samples were collected every 30–40 min twice daily from 8 cyclic and 6 pregnant cows during Days 16–20: 818 blood samples (including 216 OA and PA concurrent arterial pairs) were collected. Overall least squares means for PGF concentrations (pg/ml) in the OV, OA and PA of cyclic cows were 562, 228 and 106, respectively. A significant (P < 0·01) OA—PA difference (122pg/ml) suggests that a local transfer system, between uterine venous effluent and ovarian arterial affluent, is functional in the cow. A transfer efficiency of about 1% was estimated.
In cyclic cows differences in OA—PA concentrations of PGF were minimal on Days 16–18 and increased to about 160 pg/ml during luteal regression (Days 19–20). In pregnant cows a biphasic OA—PA pattern of difference in PGF between days was detected, with a peak on Day 18 (136 pg/ml) which was not apparent on Days 19–20. Amplitude of PGF spikes in the OA was significantly higher in cyclic (725 pg/ml) than in pregnant cows (397 pg/ml). We suggest that pregnancy suppresses PGF delivery to the ovarian circulation, resulting in maintenance of the corpus luteum in pregnant cows.
J. D. Savio, W. W. Thatcher, L. Badinga, R. L. de la Sota and D. Wolfenson
The effect of progestin and luteinizing hormone (LH) pulse frequency on dynamics of dominant follicle growth during the first follicular wave after oestrus was examined in non-lactating Holstein cows by ultrasonography. On day 8 of the cycle, cows (n = 8) received a luteolytic dose of prostaglandin F2α (PGF2α; 25 mg) and an ear implant of Norgestomet (6 mg). On day 18, cows were assigned to a crossover design in which the implants were retained (T1) or replaced with a new implant (T2). All implants were removed on day 23. After oestrus, cows underwent a normal intervening oestrous cycle. On day 8 of the third cycle, T1 and T2 were reversed among cows. Ultrasonography and blood sampling were performed on alternate days throughout the experiment. On days 10 and 19 of the third cycle, blood was sampled every 15 min for 8 h in concert with an additional control group (n = 3) sampled on day 10 of the cycle. Progesterone concentration on day 8 before PGF2α was 6.5 ± 0.5 ng ml−1. Dominance of the first wave dominant follicle was extended beyond day 18 in 15 of 16 cows for T1 and T2 periods. The original dominant follicle ovulated in five of eight T1 and none of eight T2 periods (P < 0.01). New dominant follicles were detected on day 24 ± 1 in T1 (n = 3) and on day 20.6 ± 1 in T2 (n = 8; P < 0.01) cows. Growth rate of the dominant follicle from day 8 (15.8 ± 0.4 mm) to day 18 (22.9 ± 0.6 mm) was 0.7 mm day−1 (n = 15). Preovulatory sizes for the initial and new dominant follicles were 26.8 ± 1 mm (n = 5) and 15.3 ± 0.8 mm (n = 11), respectively (P < 0.01). Intervening oestrous cycles comprised three (n = 5) and two (n = 3) follicular waves. An interaction of treatment by day (P < 0.01) for LH pulses in 8 h was detected (pulses on days 10 and 19 for T1 (5.2 ± 0.5 and 7 ± 0.7) versus T2 (5.7 ±0.5 and 3 ±0.4)). A mean of 0.7 ± 0.3 LH pulses in 8 h was detected on day 10 for control cows (n = 3). Increased LH support appears to maintain a dominant follicle, whereas high progesterone concentrations decrease LH pulse frequency leading to turnover of the dominant follicle in cattle.
S Asaf, G Leitner, O Furman, Y Lavon, D Kalo, D Wolfenson and Z Roth
Mastitis is associated with decreased fertility in dairy cows. In the current study, we created an experimental model to simulate short-term mastitis by a single intramammary administration of Gram-negative endotoxin of Escherichia coli origin (G−), or Gram-positive toxin of Staphylococcus aureus origin (G+), to examine the effect of mastitis on oocyte developmental competence. Healthy Holstein cows were synchronized, and follicular fluid (FF) of cows treated with G+ or G− and of uninfected cows (controls) was aspirated from the preovulatory follicles by transvaginal ultrasound procedure. The aspirated FF was used as maturation medium for in vitro embryo production. The distribution of matured oocytes into different cortical granule classes and meiotic stages was affected by G− administration (P<0.05) but not by G+ administration. The proportion of oocytes that cleaved to two- and four-cell stage embryos (44 h postfertilization) was lower in both G+ and G− groups than in controls (P<0.05). Blastocyst formation rate (7–8 days postfertilization) was lower in the G− group (P<0.05) and numerically lower in the G+ group compared with their uninfected counterparts. The total cell number in blastocysts did not differ among groups; however, the apoptotic index was higher in the G+ group (P<0.05), but not in the G− group, relative to controls. Examining mRNA relative abundance in oocytes and early embryos revealed mastitis-induced alterations in PTGS2 (COX2), POU5F1, and HSF1 but not in SLC2A1 (GLUT1) or GDF9. Results indicate a differential disruptive effect of mastitis induced by G− and G+ on oocyte developmental competence in association with alterations in maternal gene expression.
A Shaham-Albalancy, Y Folman, M Kaim, M Rosenberg and D Wolfenson
Low progesterone concentrations during the bovine oestrous cycle induce enhanced responsiveness to oxytocin challenge late in the luteal phase of the same cycle. The delayed effect of low progesterone concentrations during one oestrous cycle on uterine PGF(2alpha) secretion after oxytocin challenge on day 15 or 16 of the subsequent cycle was studied by measuring the concentrations of the major PGF(2alpha) metabolite (13,14-dihydro-15-keto PGF(2alpha); PGFM) in plasma. Two experiments were conducted, differing in the type of progesterone treatment and in the shape of the low progesterone concentration curves. In Expt 1, progesterone supplementation with intravaginal progesterone inserts, with or without an active corpus luteum, was used to obtain high, or low and constant plasma progesterone concentrations, respectively. In Expt 2, untreated cows, representing high progesterone treatment, were compared with cows that had low but increasing plasma progesterone concentrations that were achieved by manipulating endogenous progesterone secretion of the corpus luteum. Neither experiment revealed any differences in plasma progesterone concentrations between the high and low progesterone groups in the subsequent oestrous cycle. In both experiments, both groups had similar basal concentrations of PGFM on day 15 (Expt 1) or 16 (Expt 2) of the subsequent oestrous cycle, 18 days after progesterone treatments had ended. In both experiments, the increases in PGFM concentrations in the low progesterone groups after an oxytocin challenge were markedly higher than in the high progesterone groups. These results indicate that low progesterone concentrations during an oestrous cycle have a delayed stimulatory effect on uterine responsiveness to oxytocin during the late luteal phase of the subsequent cycle. This resulting increase in PGF(2alpha) secretion may interfere with luteal maintenance during the early stages of pregnancy.
Z Roth, R Meidan, A Shaham-Albalancy, R Braw-Tal and D Wolfenson
During the autumn, the conception rate of dairy cattle in warm countries is low although ambient temperatures have decreased and cows are no longer exposed to summer thermal stress, indicating that there may be a delayed effect of heat stress on cattle fertility. Two experiments were conducted to examine possible delayed effects of heat stress on follicular characteristics and steroid production at two distinct stages of follicular growth: medium-sized and preovulatory follicles, 20 and 26 days after heat exposure, respectively. Lactating cows were subjected to heat stress for 12 h a day in an environmental chamber, during days 2-6 of a synchronized oestrous cycle. In Expt 1, ovaries were collected on day 3 of the subsequent cycle, before selection of the dominant follicle, and medium-sized follicles were classified as atretic or healthy. In Expt 2, on day 7 of the subsequent cycle, PGF(2a) was administered and preovulatory follicles were collected 40 h later. In both experiments, follicular fluid was aspirated, granulosa and thecal cells were incubated, and steroid production was determined. In healthy medium-sized follicles (Expt 1), oestradiol production by granulosa cells and androstenedione production by thecal cells were lower (P < 0.05) and the concentration of progesterone in the follicular fluid was higher in cows that had been previously heat-stressed than in control cows (P < 0.05). In preovulatory follicles (Expt 2), the viability of granulosa cells was lower (P < 0.05) and the concentration of androstenedione in the follicular fluid and its production by thecal cells were lower (P < 0.05) in cows that had been previously heat-stressed than in control cows. In both experiments, the oestradiol concentrations in the follicular fluids were not altered by heat stress. These results demonstrate a delayed effect of heat stress on steroid production and follicular characteristics in both medium-sized and preovulatory follicles; this effect could be related to the low fertility of cattle in the autumn.
Z Roth, A Arav, A Bor, Y Zeron, R Braw-Tal and D Wolfenson
The fertility of dairy cows decreases during the summer and remains low during the cooler autumn although the animals are no longer under heat stress. The aim of this study was to characterize a delayed effect of summer heat stress on oocyte quality in the autumn and to improve oocyte quality by enhanced removal of follicles damaged during the previous summer. Lactating cows (n = 16) were subjected to heat stress during the summer. In autumn, ovarian follicles (3-7 mm in diameter) were aspirated by an ultrasound-guided procedure during four consecutive oestrous cycles. Follicles were aspirated from control cows on day 4 and from treated cows on days 4, 7, 11 and 15 of each oestrous cycle. All cows received PGF(2alpha) and GnRH injections on days 19 and 21, respectively, and maintained cyclicity, as indicated by plasma progesterone concentrations. On day 4 of each cycle, the oocytes recovered were examined morphologically, matured and activated in vitro, and cultured for 8 days. In cycle 1 (early October) both groups showed low percentages of grade 1 oocytes, cleavage, four- and eight-cell embryos, morulae and parthenogenetic blastocysts. Subsequently, the number of grade 1 oocytes increased earlier (cycle 2) in treated than in control cows (cycle 3; P < 0.05). The cleavage rate in the control group remained relatively low throughout (32-58%), whereas in the treated group it increased from 40% (cycle 1) to 75% (cycles 3 and 4; P < 0.05). The number at each stage of embryo development increased slightly but remained low throughout in the control group, whereas in the treated group significant (P < 0.05) increases of all stages were observed in cycles 3 and 4. The results show a delayed effect of summer heat stress on oocyte quality and embryo development in the autumn. Enhanced removal of the impaired cohort of follicles led to earlier emergence of healthy follicles and high quality oocytes in the autumn.
D. Wolfenson, H. Sonego, A. Shaham-Albalancy, Y. Shpirer and R. Meidan
This study, compared the endocrine function of dominant follicles of the first and second follicular waves (DF1 and DF2, respectively) and the corpora lutea that were subsequently formed. In the experiments conducted in vitro, ovaries were collected from dairy cows on day 6.1 ±0.2 or day 14.8 ±0.2 of the oestrous cycle to obtain steroidogenically active DF1 (n = 8) and DF2 (n = 7). Granulosa and thecal cells were isolated, dispersed and incubated for 16 h with testosterone (granulosa cells) or forskolin or bLH (thecal cells). Both types of cell were subsequently cultured for 9 days with forskolin and insulin. The viability of the granulosa cells was similar in DF1 and DF2, but the concentration of oestradiol in the follicular fluid was higher in DF1 than in DF2. Production of oestradiol and progesterone by granulosa cells was similar in DF1 and DF2, but androstenedione and progesterone production by thecal cells were 3.5-6.5-fold higher in DF1 than in DF2. During the 9 days of luteinization, progesterone production was similar in DF1- and DF2-derived granulosa cells, but was two- to three-fold higher in DF1- than in DF2-derived thecal cells. Experiments were also conducted in vivo. In Expt 1 in vivo, lactating cows that were assigned to ovulate DF1 or DF2 (n = 9 and 13 in replicate 1 and 2, respectively) were injected with PGF2α on days 6 and 7 or on days 14 and 15 of the oestrous cycle, respectively. A wave by replicate interaction was detected for plasma progesterone concentration in the subsequent cycle: in the first replicate, progesterone production was approximately 40% higher in cows that ovulated DF1; in the second replicate, progesterone production was similar in cows that ovulated DF1 or DF2. In Expt 2, pooled plasma progesterone in the mid-luteal phase (days 12-15) after insemination of pregnant and non-pregnant cows was approximately 30% higher in cows that had ovulated DF1 (n = 32) than in cows that had ovulated DF2 (n = 22). This study showed DF1 had a higher steroidogenic capacity compared with DF2, which may be related to the hormonal environment in which the follicles developed.