D. Wolfenson

Impaired reproduction in heat-stressed cattle: basic and applied aspects

Summer heat stress (HS) is a major contributing factor in low fertility in lactating dairy cows in hot environments. Although modern cooling systems are used in dairy farms, fertility remains low. This review summarizes the ways in which the functioning of various parts of the reproductive system of cows exposed to HS is impaired. The dominance of the large follicle is suppressed during HS, and the steroidogenic capacity of theca and granulosa cells is compromised. Progesterone secretion by luteal cells is lowered during summer, and in cows subjected to chronic HS, this is also reflected in lower plasma progesterone concentration. HS has been reported to lower plasma concentration of LH and to increase that of FSH; the latter was associated with a drastic reduction in plasma concentration of inhibin. HS impairs oocyte quality and embryo development, and increases embryo mortality. High temperatures compromise endometrial function and alter its secretory activity, which may lead to termination of pregnancy. In addition to the immediate effects, delayed effects of HS have been detected as well. Among them, altered follicular dynamics, suppressed production of follicular steroids, and low quality of oocytes and developed embryos. These may explain the low fertility of cattle during the cool autumn months. Hormonal treatments improve low summer fertility to some extent but not sufficiently for it to equal winter fertility. A limiting factor is the inability of the high-yielding dairy cow to maintain normothermia. A hormonal manipulation protocol, which induces timed insemination, has been found to improve pregnancy rate and to reduce the number of days open during the summer.

Effect of environmental heat stress on follicular development and steroidogenesis in lactating Holstein cows.

Lactating Holstein cows were utilized over two replicate periods (July and September, 1990) to examine the effect of summer heat stress on follicular growth and steroidogenesis. On day of synchronized ovulations, cows were assigned to shade (n=11) or no shade (n=12) management systems. Follicular development was monitored daily by ultrasonography until ovariectomy on Day 8 post estrus. At time of ovariectomy, dominant and second largest follicles were dissected from the ovary. Aromatase activity and steroid concentrations in dominant and subordinate follicles were measured. Acute heat stress had no effects on patterns of growth of first wave dominant and subordinate follicles between Days 1 and 7 of the cycle. Compared with shaded cows, the heat stressed cows did not have suppression of medium size (6 to 9 mm) follicles between Days 5 and 7. A treatment x follicle interaction was detected (P<0.01) for follicular diameter and fluid volume at Day 8. Dominant follicles in shade were bigger (16.4>14.5 mm) and contained more fluid (1.9>1.1 ml) than dominant follicles in no shade. Conversely, subordinate follicles in no shade were bigger (10.1>7.9 mm) and contained more fluid (0.4>0.2 ml) than subordinate follicles in shade. Concentrations of estradiol in plasma and follicular fluid were higher (P<0.01) in July than in September. Heat stress appears to alter the efficiency of follicular selection and dominance, and to have adverse effects on the quality of ovarian follicles.

Seasonal and acute heat stress effects on steroid production by dominant follicles in cows

The present study concerned the seasonal and acute effects of heat stress on steroid concentrations in follicular fluid and on steroid production by granulosa and theca interna cells, in bovine dominant follicles. Three groups of cows were studied: summer (n = 5), autumn (n = 5) and winter (n = 9) cows. During the winter season, another group of cows was acutely heat-stressed from days 3 through 5 of the estrous cycle (n = 5). On day 7 of the estrous cycle, follicular fluid from first-wave dominant follicles was aspirated, and dispersed granulosa and theca cells from each seasonal group were incubated for 18 h at normothermic (37.5 degrees C) or high (40.5 degrees C) temperatures. Cells were incubated in media only or in media containing testosterone (300 ng ml-1, for granulosa cells) or forskolin (4 micrograms ml-1, for theca cells). In follicular fluid the 17 beta-estradiol concentration was high (P < 0.05) in winter and low in autumn, and summer, the androstenedione concentration was high in summer (P < 0.05), low in autumn, and intermediate in winter. During the winter season, acute in vivo heat stress increased follicular fluid androstenedione and decreased estradiol to levels comparable with those prevailing in summer. Basal and forskolin-stimulated androstenedione production by theca cells was higher (P < 0.05) in the winter group than in the summer and autumn groups, and also higher than in the cows that were heat-stressed during winter, which suggests that theca cell function is susceptible to chronic (summer), short-term (winter) and delayed (autumn) heat stresses. In vitro incubation at high temperature (40.5 degrees C) reduced the high, forskolin-stimulated androstenedione production in winter (P < 0.05). Estradiol production by granulosa cells was high in winter and autumn, and low in summer (P < 0.05). Acute heat stress in winter did not alter estradiol production relative to winter controls, whereas a high incubation temperature (40.5 degrees C) reduced (P < 0.05) estradiol production only in the autumn, when the highest production rate was recorded. The results indicate a differential effect of heat stress on the functions of granulosa and theca cells. Both concurrent and delayed effects of heat stress on the steroidogenic capacity of ovarian follicles in cattle are presented.

Heat stress effects on capillary blood flow and its redistribution in the laying hen

The effect of heat stress on capillary blood flow (CBF) distribution was examined in laying hens, using 15 micron microspheres, by determining CBF before and after elevating body temperature by 1–2°C. No change was evident in unfeathered metatarsal skin, although its temperature increased by 7°C. Breast skin CBF change was 3.5 times larger than that of back skin. Comb CBF increase was larger than in wattles. CBF in upper respiratory tract increased proportionally to increment in respiratory frequency. Digestive system CBF was reduced by hyperthermia: the effect was pronounced in its upper organs (46% of normal) and decreased along the tract. CBF increased 4-fold in an expiratory abdominal muscle, a smaller rise occurred in a pectoral muscle and no change in a leg muscle. CBF in the tibia fell to 64% of normal. In the reproductive system, CBF fell to 58% of control level in the uterus, to 70–80% in the larger ovarian follicles and infundibulum with no significant changes in magnum and isthmus. Cerebral CBF increased during hyperthermia.Heat stress significantly reduced CBF to inner body organs, with marked differences between systems as well as within systems. Changes were more pronounced on 2°C hyperthermia than on 1°C hyperthermia.

Administration of Prostaglandin F2α During the Early Bovine Luteal Phase Does Not Alter the Expression of ET-1 and of Its Type A Receptor: A Possible Cause for Corpus Luteum Refractoriness

Abstract Luteal regression is initiated by prostaglandin F2α (PGF2α). In domestic species and primates, demise of the corpus luteum (CL) enables development of a new preovulatory follicle. However, during early stages of the cycle, which are characterized by massive neovascularization, the CL is refractory to PGF2α. Our previous studies showed that endothelin-1 (ET-1), which is produced by the endothelial cells lining these blood vessels, plays a crucial role during PGF2α-induced luteolysis. Therefore, in this study, we compared the effects of PGF2α administered at the early and mid luteal phases on ET-1 and its type A receptors (ETA-R) along with plasma ET-1 and progesterone concentrations, and the mRNA levels of PGF2α receptors (PGF2α-R) and steroidogenic genes. As expected, ET-1 and ETA-R mRNA levels were markedly induced in midcycle CL exposed to luteolytic dose of PGF2α analogue (Cloprostenol). In contrast, neither ET-1 mRNA nor its receptors were elevated when the same dose of PGF2α analogue was administered on Day 4 of the cycle. In accordance with ET-1 expression within the CL, plasma ET-1 concentrations were significantly elevated 24 h after PGF2α injection only on Day 10 of the cycle. The steroidogenic capacity of the CL (plasma progesterone as well as the mRNA levels of steroidogenic acute regulatory protein and cytochrome P450scc) was only affected when PGF2α was administered during midcycle. Nevertheless, PGF2α elicited certain responses in the early CL: progesterone and oxytocin secretion were elevated, and PGF2α-R was transiently affected. Such effects probably result from PGF2α acting on luteal steroidogenic cells. These findings may suggest, however, that the cell type mediating the luteolytic actions of PGF2α, possibly the endothelium, could yet be nonresponsive during the early luteal phase.

The effect of a GnRH analogue on the dynamics of follicular development and synchronization of estrus in lactating cyclic dairy cows

A GnRH analogue was used to synchronize ovarian follicular development prior to an injection of PGF(2alpha) for the synchronization of estrus in lactating Holstein cows. On Day 12 (estrus = Day 0) of the experimental cycle, cows (n = 8) were injected with 8 mug Buserelin (BUS group), followed by 25 mg PGF(2alpha) 7 d later (Day 19). Control cows (n = 7) received PGF(2alpha) on Day 12 (PGF group). Ovaries were scanned daily via ultrasonography, and plasma progesterone and estradiol concentrations were determined. Sizes of all visible follicles were recorded. Follicles were classified as small (3 to 5 mm), medium (6 to 9 mm), or large (> or = 10 mm). Between Days 12 and 16 of the cycle, the number of large follicles in PGF cows remained unchanged (1.2), whereas in the BUS group, the number of large follicles decreased from 1.3 on Day 12 to 0.5 on Day 15. Only 4 of 7 PGF cows ovulated a second-wave dominant follicle. In the BUS group, 7 of 8 cows ovulated a GnRH analogue induced dominant follicle that was first identified on Day 15. During the follicular phase (last 5 d prior to estrus), plasma progesterone declined in association with CL regression in both groups, and estradiol concentrations increased, reaching higher (P<.0.05) preovulatory peak concentration in BUS cows than in PGF cows (14.0 +/- 1.0 vs 10.4 +/- 1.1 pg/ml). The number of medium-size follicles was smaller and the number of small-size follicles tended to be higher in BUS cows than in the PGF-treated group. On the day of estrus, the size of the ovulatory follicle (16.1 vs 13.3 mm) and the size difference between the ovulatory and second largest follicle (11.4 vs 6.2 mm) were both larger in BUS cows than in PGF-treated cows, suggesting a more potent dominance effect of the ovulatory follicle in the BUS cows. This study suggests that a GnRH analogue can alter follicular development prior to synchronization of estrus with an injection of PGF(2alpha) in lactating dairy cows.

Antioxidant capacity is correlated with steroidogenic status of the corpus luteum during the bovine estrous cycle

The reactions of steroid hormone biosynthesis are accompanied by formation of oxygen radicals. We determined the levels of some antioxidants and antioxidative enzymes at different developmental stages of bovine corpora lutea to examine their correlation with steroidogenic status. Plasma progesterone concentrations of estrous cycle synchronized cows increased until day 16, and then decreased rapidly during luteal regression. The levels of steroidogenic cytochrome P450scc and adrenodoxin paralleled the changes in plasma progesterone. Among the antioxidative enzymes examined, the SOD and catalase activities showed patterns most similar to plasma progesterone. Catalase and SOD activities increased 6-8 fold from day 6 to 16 of the estrous cycle and then decreased during the luteal regression. Ascorbate and beta-carotene showed low but significant correlation with P450scc and plasma progesterone levels. The profiles of two lipophilic antioxidants in corpora lutea were very different. beta-carotene concentration increased by approximately 6 fold from day 6 to 16, and decreased in regressive tissue. alpha-tocopherol showed a 3 fold increase between days 6 and 9 followed by a rapid decrease. Thus, at the peak of steroidogenesis at mid-luteal phase alpha-tocopherol levels decreased, but beta-carotene levels increased. The correlation between the levels of some antioxidant enzymes and compounds with progesterone levels indicates that antioxidative mechanisms are activated to cope with steroidogenesis dependent oxyradical formation in the bovine corpus luteum.

Effect of season on follicular dynamics and plasma concentrations of estradiol-17β, progesterone and luteinizing hormone in lactating Holstein cows

Lactating Holstein cows (n=16), averaging 64.1 d in milk, were utilized over 4 replicate months (April, June, August and November) in a shade management system to examine the effects of season on follicular dynamics and plasma concentrations of estradiol (E2), progesterone (P4) and luteinizing hormone (LH). Cows were synchronized to estrus using a combination of Buserelin (GnRH, 8 ug) and prostaglandin F2α (PGF2α, 25 mg) given 7 d apart. Follicular development was monitored daily by ultrasonography, and plasma concentrations of E2, P4 and LH measured by radioimmunoassay. The replicate month had no detectable effects on estrus interval (3.1 ± 0.3 d) or percentage of cows (78.1 ± 9.4%) that expressed estrus following GnRH and PGF2α treatment. Preovulatory follicles grew at faster rates (P<0.01) in June (2.0 ± 0.6 mm/d), than in April (1.1 ± 0.6 mm/d), August (1.0 ± 0.6 mm/d) or November (1.2 ± 0.6 mm/d). First wave dominant follicles were consistently larger in April than in June, August and November. The larger and more persistent size of the first wave dominant follicle in April was associated with an earlier regression of the largest subordinate follicle and a sharper decrease in the number of medium size follicles (6 to 9 mm) by Day 9 of the estrous cycle. Conversely, growth of the first wave dominant follicle was slower and the largest subordinate follicle was more persistent in August than in April, June or November. The proestrous rise in plasma E2 occurred faster (P<0.01) in August (10.1 ± 2.1 pg/d) than in April (4.6 ± 2.1 pg/d), June (5.3 ± 2.1 pg/d) or November (5.9 ± 2.1 pg/d). Concentrations of P4 in plasma increased and reached higher (P<0.01) luteal values in August (15.1 ± 0.6 ng/ml) and November (16.0 ± 0.6 ng/ml) than in April (6.1 ± 0.6 ng/ml) and June (10.6 ± 0.6 ng/ml). There was no detectable effect of month on LH pulse characteristics 48 h post-PGF2α. The maximum size of the corpus luteum (CL) was greatest in November and was related positively to diameter of the ovulatory follicle of the preceding cycle. Results indicated that ovarian follicular development and dominance may be altered during summer months. However, it is uncertain whether these changes can be related to the well-documented low breeding efficiency during warmer months of the year in subtropical environments.

Secretion of PGF2α and oxytocin during hyperthermia in cyclic and pregnant heifers

The effects of acute heat stress (HS) and oxytocin (OT) injection on plasma concentrations of PGF2alpha and OT were examined in cyclic (C; n = 15) and pregnant (P; n = 11) dairy heifers. On Day 17 of synchronized estrous cycles, animals were randomly assigned to either thermoneutral (TN; 20 degrees C, 20% RH) or HS (42 degrees C, 60% RH) chambers. The jugular vein of each heifer was cannulated and blood samples collected hourly for 4 h, then every 15 min for an additional 3 h. Oxytocin (100 IU) was injected (IV) 5 h after the start of blood collection. Plasma samples were assayed subsequently for concentrations of 13,14-dihydro-15-keto PGF2alpha (PGFM) and OT. During the 7-h experiment, body temperature of HS heifers reached 41.2 degrees C as compared to 38.5 degrees C in control heifers. Plasma concentrations of PGFM increased (P<0.05) and peaked 30 min after OT injection in C (890 pg/ml) and P (540 pg/ml) heifers. In C heifers, heat stress failed to alter PGFM concentrations either before or after OT injection. In the P group, PGFM concentrations following OT injection tended to be higher in HS heifers were further TN heifers (peak values of 690 vs. 410 pg/ml). Pregnant TN and HS heifers were further classified as responders or non-responders to OT challenge according to a cutoff value for PGFM of 193 pg/ml (overall mean of C heifers minus 1 SD). Five of six HS and one of five TN pregnant heifers were classified as responders (P<0.06). Oxytocin concentrations in plasma prior to injection of exogenous OT were not affected by HS or pregnancy status. It is concluded that in C heifers, acute HS in vivo does not cause any further rise in PGF2alpha secretion. However, in P heifers, HS appears to antagonize suppressive effects of the embryo on uterine secretion of PGF2alpha, as indicated by the larger proportion of P heifers responding to OT challenge.

Exposure to endotoxin during estrus alters the timing of ovulation and hormonal concentrations in cows

The effect of intramammary (IMM) or intravenous (IV) administration of E. coli endotoxin (LPS), at the onset of estrus, at the time of ovulation was examined. Steroid and gonadotropin concentrations around ovulation were also determined. Lactating Holstein cows (n=33) were assigned to saline-controls (n=12) and treated with LPS-IV (0.5 microg/kg; n=13) or LPS-IMM (10 microg; n=8). Synchronized cows were observed continuously for estrus. LPS (or saline) was injected within 30 min from the onset of standing estrus, at peak estradiol concentrations. The typical rise of body temperature, somatic cell count, cortisol, and NAGase activity was noted. One-third of both LPS-IV- and LPS-IMM-treated cows were manifested by an extended estrus to ovulation (E-O) interval of around 75 h or did not ovulate, compared with about 30 h in the other 2/3 of LPS cows and all controls. Estradiol concentrations 24 h before and after LPS did not differ between groups. However, LPS-IV cows with extended intervals exhibited another estrus and an additional rise of estradiol followed by delayed ovulation. LPS-treated cows with a delayed E-O interval had low or delayed LH surge; two LPS-treated cows did not exhibit LH surge and did not ovulate. All control cows exhibited normal hormone levels. Delayed ovulation was associated with a delayed rise of luteal progesterone. The results indicated that exposing cows to endotoxin during estrus induced a decreased and delayed LH surge in one-third of the cows. This was associated with delayed ovulation, which reduces the chances of successful fertilization.