L. Badinga

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.

Effects of growth hormone and insulin-like growth factor-I on development of in vitro derived bovine embryos

The objectives of this study were to determine whether the addition of growth hormone (GH) to maturation medium and GH or insulin-like growth factor-I (IGF-I) to culture medium affects development of cultured bovine embryos. We matured groups of 10 cumulus-oocyte complexes (COCs) in serum-free TCM-199 medium containing FSH and estradiol with or without 100 ng/ml GH. After fertilization, we transferred groups of 10 putative zygotes to 25 microl drops of a modified KSOM medium containing the following treatments: non-specific IgG (a control antibody, 10 microg/ml); GH (100 ng/ml) + IgG (10 microg/ml, GH/IgG); IGF-I (100 ng/ml) + IgG (10 microg/ml, IGF/IgG); antibody to IGF-I (10 microg/ml, anti-IGF); GH (100 ng/ml) + anti-IGF (10 microg/ml GH/anti-IGF); IGF-I (100 ng/ml) + anti-IGF (10 microg/ml, IGF/anti-IGF); no further additions (control). We repeated the experiment six times. Adding GH to the maturation medium increased cleavage rates at Day 3 compared to control (87.3 +/- 1.2% > 83.9 +/- 1.2%; P < 0.05) but had no effects on blastocyst development at Day 8. At Day 8, blastocyst development was greater (P < 0.01) for GH/IgG (24.8 +/- 2.5%) and IGF/IgG (33.7 +/- 2.5%) than for IgG (16.1 +/- 2.1%) and greater for IGF/IgG than for GH/IgG (P < 0.02). Blastocyst development at Day 8 did not differ between anti-IGF (20.4 +/- 1.8%) and GH/anti-IGF (24.1 +/- 1.9%) or IGF/anti-IGF (17.7 +/- 1.9%), but it was greater for GH/anti-IGF than for IGF/anti-IGF (P < 0.05). The Day 8 blastocysts of GH/IgG and IGF-I/IgG groups had a higher (P < 0.01) number of cells than the IgG group. The addition of anti-IGF-I eliminated the effects of IGF-I on cell number but did not alter GH effects. In conclusion, both GH and IGF-I stimulate embryonic development in cattle and GH effects may likely involve IGF-I-independent mechanisms.

Polyunsaturated Fatty Acids and Bovine Interferon-τ Modify Phorbol Ester-Induced Secretion of Prostaglandin F2α and Expression of Prostaglandin Endoperoxide Synthase-2 and Phospholipase-A2 in Bovine Endometrial Cells

Abstract Embryonic mortality in cattle may occur because of inadequate inhibition of uterine secretion of prostaglandin (PG) F2α mediated by bovine interferon-τ (bIFN-τ). The objectives of the present study were to determine whether polyunsaturated fatty acids inhibit secretion of PGF2α from bovine endometrial cells induced by stimulating protein kinase C with phorbol 12,13 dibutyrate (PDBu) and to investigate possible mechanisms of action. Confluent cells were exposed for 24 h to 100 μM of linoleic, arachidonic (AA; C20:4, n-6), linolenic (LNA; C18:3, n-3), eicosapentaenoic (EPA; C20:5, n-3), or docosahexaenoic (DHA; C22:6, n-3) acid. After incubation, cells were washed and stimulated with PDBu. The EPA, DHA, and LNA attenuated secretion of PGF2α in response to PDBu. The EPA and DHA were more potent inhibitors than LNA. The EPA inhibited secretion of PGF2α at 6.25 μM. Secretion of PGF2α in response to PDBu decreased with increasing incubation time with EPA. Both bIFN-τ and EPA inhibited secretion of PGF2α, and their inhibitory effects were additive. The bIFN-τ, but not EPA, reduced the abundance of PG endoperoxide synthase-2 (PGHS-2) mRNA. Incubation with 100 μM EPA, DHA, or AA for 24 h followed by treatment with PDBu did not affect concentrations of PGHS-2 and phospholipase A2 proteins. The EPA and DHA inhibit secretion of PGF2α through a mechanism different from that of bIFN-τ. The effect of EPA on PGF2α secretion may be caused by competition with AA for PGHS-2 activity or reduction of PGHS-2 activity. The use of EPA and DHA to inhibit uterine secretion of PGF2α and to improve embryonic survival in cattle warrants further investigation.

Bovine somatotropin increases embryonic development in superovulated cows and improves post-transfer pregnancy rates when given to lactating recipient cows.

Previous studies indicated that the use of bovine somatotropin (bST) in concurrence with a timed artificial insemination (TAI) protocol increased pregnancy rates. However, the mechanisms for such a bST effect on fertility were not clear. Objectives of this study were to determine the effects of bST on fertilization and early embryonic development after cows received a superovulation treatment, test whether embryos recovered from bST-treated cows were more likely to survive after transfer to recipients, and evaluate whether treatment of recipient cows with bST affects pregnancy rates. Lactating (n = 8) and nonlactating (n = 4) Holstein donor cows were superovulated, inseminated at detected estrus and assigned to a nontreated control group or to a treatment group receiving a single injection of bST (500 mg, sc) at insemination. Embryos were nonsurgically flushed 7 days after AI and frozen in ethylene glycol for direct transfer. Embryos derived from bST-treated (bST-embryos) or control (control-embryos) donors were transferred to lactating Holstein recipient cows that received either bST treatment 1 day after estrus (500 mg, sc; bST-recipients) or were untreated controls (control-recipients). Thus, there were four treatment groups: control-embryos/control-recipients (n = 43), bST-embryos/control-recipients (n = 41), control-embryos/bST-recipients (n = 37), and bST-embryos/bST-recipients (n = 60). Pregnancy was determined by palpation per rectum 33-43 days after embryo transfer. Unfertilized ova per flush was less for bST than for control (1.0 +/- 0.9 < 3.7 +/- 0.9; P < 0.04). Percentage of transferable embryos was greater for bST than for control (77.2% > 56.4%; P < 0.01). Number of blastocysts per flush was greater for bST than for control (2.4 +/- 0.7 > 0.4 +/- 0.7; P < 0.04). Pregnancy rates following embryo transfer were 25.6% for control-recipient/control-embryo, 43.2% for bST-recipient/control-embryo, 56.1% for control-recipient/bST-embryo, and 43.3% for bST-recipient/bST-embryo. Transfer of bST-embryos increased pregnancy rates compared with transfer of control-embryos (P < 0.04). An interaction between embryo and recipient treatments (P < 0.05) indicated that treatment of recipient cows with bST increased pregnancy rates as compared to control-recipients that received a control-embryo. However, there was no additive effect when bST-recipients received a bST-embryo. Administration of bST at AI decreased the number of unfertilized ova, increased the percentage of transferable embryos, and stimulated embryonic development to the blastocyst stage. Moreover, bST affected both early embryonic development and recipient components to increase pregnancy rates following embryo transfer.

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.

Interferon-τ Modulates Phorbol Ester-Induced Production of Prostaglandin and Expression of Cyclooxygenase-2 and Phospholipase-A2 from Bovine Endometrial Cells

Abstract Antiluteolytic actions of bovine interferon-tau (bIFN-τ) require suppression of prostaglandin F2α (PGF2α) production. Our objective was to test whether bIFN-τ could block PGF2α production and synthesis of phospholipase A2 (PLA2) and cyclooxygenase-2 (COX-2) enzymes induced by a protein kinase C (PKC) stimulator (phorbol 12,13 dibutyrate; PDBu). Bovine endometrial epithelial (BEND) cells were treated with PDBu in the presence or absence of bIFN-τ. Medium samples were analyzed for concentrations of PGF2α, whole-cell extracts were analyzed for abundance of PLA2 and COX-2 by immunoblotting, and RNA extracts were examined for steady-state levels of COX-2 mRNA by Northern blotting. The PDBu stimulated production of PGF2α between 3 and 12 h, levels of COX-2 mRNA by 3 h and protein expression of COX-2 and PLA2 by 6 and 12 h, respectively. Added concomitantly with PDBu, bIFN-τ suppressed PGF2α production, steady-state levels of COX-2 mRNA, and expression of COX-2 and PLA2 proteins. Added after a 3-h stimulation with PDBu alone, bIFN-τ suppressed PGF2α production after 1 h. Bovine IFN-τ inhibited intracellular mechanisms responsible for PGF2α production in BEND cells, and this could be through both cytosolic and nuclear actions.

EFFECTS OF SOMATOTROPIN ON THE CONCEPTUS, UTERUS, AND OVARY DURING MATERNAL RECOGNITION OF PREGNANCY IN CATTLE

Effects of recombinant bovine somatotropin (rbST) on ovarian and uterine function and the production of components of the insulin-like growth factor (IGF) system were examined during the period of maternal recognition of pregnancy in cattle. Lactating dairy cows were treated with 25 mg/d rbST (n = 8) or saline (n = 8) for 16 d after estrus. Ovaries, uteri, and conceptuses were collected on Day 17 after estrus. The length (millimeters) of the conceptus was recorded. The concentration of IGF-I and the content of IGF-binding proteins (BP) in uterine flushings were determined. Corpora lutea (CL) were weighed, and the number of follicles (> or = 2 mm in diameter) were counted. Follicular fluid from the largest and second-largest follicles was assayed for the concentration of IGF-I, IGFBP, progesterone, and estradiol. The length of the conceptus and the total amount of IGF-I in uterine fluid were similar for rbST and control. Recombinant bST increased 1) the weight of the CL, 2) the number of largest follicles (10 to 15 mm in diameter), 3) the concentration of IGF-I in the follicular fluid, 4) the follicular fluid content of IGFBP of the largest estrogenic follicle, and 5) the quantity of IGFBP in uterine flushings. The concentration of progesterone in the follicular fluid tended to be increased in rbST-treated cows, whereas the concentration of estradiol was similar to that of control cows. The concentration of progesterone in plasma was similar for rbST compared with control. In conclusion, the administration of rbST in lactating dairy cows for 16 d after estrus did not alter the growth of the conceptus collected on Day 17. The greatest responses to rbST were found within the ovary, where rbST increased the weight of the CL and altered the amount of IGF-I and IGFBP in the follicular fluid.

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.

Dynamics of ovarian follicular development in cattle following hysterectomy and during early pregnancy

Three experiments were conducted to evaluate ovarian follicular dynamics and functional activity during pregnancy in cattle. In 11 pregnant Charolais cows of Experiment I, size of largest follicle, number of follicles and accumulated follicle size were reduced by day 27 of pregnancy on the ovary bearing the corpus luteum (CL) but not on the non-CL bearing ovary. In experiment II, local attenuation of ovarian follicular development on the CL bearing ovary of seven pregnant heifers was evident compared to the contralateral ovary without the CL. However, in four hysterectomized heifers, follicular development was sustained on both the CL- and non-CL bearing ovaries when CL maintenance was achieved without presence of the uterus or conceptus. In Experiment III, steroidogenic characteristics of the largest and second largest follicles at 17 d postestrus were evaluated for seven pregnant and six cyclic cattle. Follicle by physiological status interactions were detected for both aromatase activity of the follicle and follicular fluid concentrations of estradiol and progesterone. In cyclic cows, the largest follicle had appreciably more aromatase activity than did the second largest follicle; whereas, aromatase activity of the largest follicle from pregnant cows was less than that of cyclic cows. However, in pregnant cows the second largest follicle became the estrogen-active follicle, and this follicle occurred with a higher frequency on the ovary contralateral to the CL-bearing ovary. These changes in aromatase activity were reflected by parallel changes in estrogen concentrations of follicular fluid. The higher progesterone concentration in follicular fluid of the largest follicle in pregnant cows provided further confirmation of their atretic status. In conclusion, during early pregnancy the conceptus and/or uterus ipsilateral to the conceptus appear to secrete compounds which alter local follicular steroidogenic activity and attenuate subsequent follicular growth between 17 to 34 d of pregnancy on the CL-bearing ovary. This local mechanism acting within the ovary may contribute to the antiluteolytic effects of early pregnancy in cattle.

The Porcine Insulin-Like Growth Factor System: At the Interface of Nutrition, Growth and Reproduction

The IGF system is implicated in the regulation of cellular response to protein- and energy-restriction. Although it is clear that the IGF and their binding proteins are profoundly influenced by dietary factors, a number of important questions remain about this relationship. In particular, although studies to date have focused on nutritional modulation of hepatic IGF gene expression, the molecular mechanisms underlying metabolic regulation of liver IGF and IGF binding protein genes remain relatively unknown. Moreover, the potential effects of altered nutrition on the expression and/or actions of IGF system components in tissues other than the liver have been examined only in cursory fashion. Many of these studies have used rats, an admittedly important model, but one which differs from the human in a potentially significant way: rats lack circulating IGF-II and IGFBP-2 during post-weaning and adult life. Here, we summarize current research on the porcine IGF system and highlight the particular usefulness this system may offer for unraveling the complex relationships of nutrition and systemic/local IGF expression and actions that are relevant to human nutritional physiology.