Comparing effects of bovine Streptococcus and Escherichia coli mastitis on impaired reproductive performance.

In 2 epidemiological studies, we evaluated the effect of mastitis induced by gram-positive Streptococcus and gram-negative Escherichia coli on impaired reproductive performance in lactating Holstein cows. In the first study, 52,202 cows from 178 dairy farms throughout Israel were divided into groups based on infection before first artificial insemination (AI) with Streptococcus or E. coli, 3 groups with elevated somatic cell count (SCC) without infection by those pathogens [low SCC (200-400) × 103 cell/mL; medium SCC (401-1,000) × 103 cell/mL; high SCC, >1,000 × 103 cell/mL], and uninfected controls. Pregnancy per first AI (P/1stAI) and pregnancy rate at 300 d in milk (PREG 300) were analyzed by the GLIMMIX procedure (SAS); number of AI per pregnancy (AI/P), days open, and rest days (calving to first AI) were analyzed by the MIXED procedure (SAS Institute Inc., Cary, NC). Values of P/1stAI were similarly low for Streptococcus and E. coli (27-28%) versus 42% in controls; PREG 300 was lower for Streptococcus (76%) than for E. coli (79%) versus 88% for uninfected controls and a mean 83% for the elevated SCC groups. Days open and number of AI/P were higher than in controls and similar in Streptococcus and E. coli groups. The second study included 778 cows on 6 dairy farms; the cows were infected before first AI by Streptococcus or E. coli or uninfected. Resumption of cyclicity was determined by an automated activity-monitoring system, and data were sorted by time of infection before or after cyclicity resumed. The Streptococcus group had lower P/1stAI before and after cyclicity (26 and 27%, respectively) than the E. coli group (31 and 34%, respectively) and uninfected controls (42%). Notably, PREG 300 in the Streptococcus group before (73%) and after (67%) cyclicity was much lower than for the E. coli group (85 and 93%, respectively) and the controls (95%). A marked rise in day of cyclicity resumption (∼80 d) was observed in cows that were infected early on. Number of AI/P was higher in the mastitic groups than in uninfected controls. Uterine disease postpartum, although more prevalent among Streptococcus cows, did not substantially alter the larger reduction in P/1stAI and PREG 300 in Streptococcus versus E. coli cows. Thus, long-term Streptococcus-induced mastitis disrupted fertility more than short-term acute E. coli-induced mastitis, resulting in a much higher percentage of Streptococcus cows in late lactation that did not conceive due to reproduction failure.

Comparing the effects of heat stress and mastitis on ovarian function in lactating cows: basic and applied aspects.

Reduced reproductive performance of lactating cows is strongly associated with environmental and pathogenic stressors. This review summarizes the most recent knowledge on the effects of acute or chronic heat stress (HS) and acute or chronic intramammary infection (IMI) on ovarian function. It also offers various approaches for improving the fertility of cows under chronic HS or IMI. Comparing the 2 stressors reveals a few similarities in the mode of alteration in the hypothalamus-pituitary-ovarian axis, in particular, in the follicle and its enclosed oocyte. Both HS and IMI cause a reduction in the preovulatory LH surge, with a pronounced effect in cows with IMI, and consequently, ovulation is being delayed or inhibited. Both stresses induce changes in follicular growth dynamics, reduce follicular steroidogenesis, and disrupt follicular dominance. Unlike their effects on follicular function, the effects of mastitis and HS on corpus luteum (CL) function are debatable. Under chronic summer thermal stress, several, but not all, studies show reduced progesterone secretion by the CL. Subclinical mastitis does not affect CL function, whereas the effect of clinical mastitis is controversial; some show a reduction in progesterone, whereas others do not. Both stresses have been found to impair cytoplasmic and nuclear maturation of oocytes, associated with reduced embryonic development. These findings have provided insights into the mechanism by which HS and IMI compromise fertility, which enable developing new strategies to mitigate these effects. For instance, treatment with GnRH and PGF2α to induce follicular turnover successfully improved conception rate in subpopulations of HS cows during the summer, in particular, primiparous cows and cows with high BCS. The "Ovsynch" program, also based on the use of GnRH and PGF2α, has been shown to improve conception rate of subclinical mastitic cows, most likely due to better synchronization of timing of ovulation with that of AI. Supplementing progesterone after AI improves conception rate of HS cows, particularly those with postpartum uterine disease and low BCS. It should be noted that similarities between the 2 stressors do not necessarily suggest a shared mechanism. Although not clear enough, an additive deleterious effects of HS and IMI on reproduction is suggested.

Two approaches to improve fertility of subclinical mastitic dairy cows.

Mastitis, particularly in its subclinical form, is a widely spread disease that reduces the fertility of lactating cows. A major cause of poor conception risk has been associated with delayed ovulation of a large subgroup of subclinical mastitic cows. This study examined 2 approaches to improve fertility in this subgroup. Subclinical mastitic cows were defined by somatic cell count elevated above a threshold of 150,000 cells/mL of milk determined in all monthly test day samples collected before AI. Uninfected (control) cows were defined by somatic cell count below threshold. In experiment 1, we examined a hormonal approach aimed to correct the timing of ovulation in mastitic cows in which it would otherwise be delayed. The probability of conception of mastitic and uninfected groups following Ovsynch (OVS) and timed AI versus AI following detected estrus (E) was examined (n=1,553 AI) and analyzed by a multivariable, logistic model statement using the GLIMMIX procedure of SAS. The OVS protocol significantly elevated the probability of conception of mastitic cows to a level similar to that of their uninfected counterparts. Actual mean conception risks for uninfected-E, subclinical-E, uninfected-OVS, and subclinical-OVS groups were 41.8, 26.4, 39.3, and 40.5%, respectively. The OVS protocol did not improve probability of conception in cows diagnosed with uterine disease postpartum. In experiment 2, a management approach aimed to better synchronize timing of ovulation with timing of AI in subclinical mastitic cows was examined. A second AI was added 24h after the first (routine) AI, following detection of natural estrus. Probability of conception did not differ between subclinical mastitic cows inseminated once or twice. Lack of improvement in conception risk might be related to low preovulatory LH surge in mastitic cows, which is likely to induce not only delayed ovulation but also disruption of oocyte maturation. Thus the OVS protocol can improve fertility of subclinical mastitic cows, probably due to "corrected" timing of ovulation in cows in which it would otherwise be delayed.

Subclinical mastitis disrupts oocyte cytoplasmic maturation in association with reduced developmental competence and impaired gene expression in preimplantation bovine embryos.

Subclinical chronic mastitis was induced to examine the effects on oocyte developmental competence. Uninfected Holstein cows were intramammary administrated with serial (every 48h for 20 days) low doses of toxin of Staphylococcus aureus origin (Gram-positive; G+), endotoxin of Escherichia coli origin (Gram-negative; G-) or sterile saline (control). Follicular fluid of toxin- and saline-treated cows was aspirated from preovulatory follicles and used as maturation medium. Oocytes harvested from ovaries collected at the abattoir were matured and then fertilised and cultured for 8 days. The percentage of oocytes undergoing nuclear maturation, determined by meiotic nuclear stages, did not differ between groups. Cytoplasmic maturation, determined by cortical granule distribution, was affected by both toxins (PPPPTGS2) mRNA increased, whereas that of growth differentiation factor 9 (GDF9) decreased in matured oocytes. In addition, PTGS2 expression increased and POU class 5 homeobox 1 (POU5F1) expression decreased in 4-cell embryos developed from both G+ and G- oocytes. Thus, regardless of toxin type, subclinical mastitis disrupts oocyte cytoplasmic maturation and alters gene expression in association with reduced developmental competence.

Hormonal treatment before and after artificial insemination differentially improves fertility in subpopulations of dairy cows during the summer and autumn.

Reduced conception rate (CR) during the hot summer and subsequent autumn is a well-documented phenomenon. Intensive use of cooling systems can improve summer and autumn reproductive performance, but is unable to increase CR to winter and spring levels. We examined whether combined hormonal treatments--to increase follicular turnover before artificial insemination (AI) and progesterone supplementation post-AI--might improve fertility of cooled cows during the summer and autumn. The experiment was conducted from July to November in 3 commercial herds in Israel and included 707 Holstein cows at 50 to 60 d in milk (DIM). Cows were hormonally treated to induce 2 consecutive 9-d cycles, with GnRH administration followed by PGF2α injection 7 d later, followed by an intravaginal insert containing progesterone on d 5 ± 1 post-AI for 14 d. Both untreated controls (n=376) and treated cows (n=331) were inseminated following estrus, and pregnancy was determined by palpation 42 to 50 d post-AI. First-AI CR data revealed a positive interaction between treatment and cows previously diagnosed with postpartum uterine disease [odds ratio (OR) 2.24]. Interaction between treatment and low body condition score tended to increase the probability of first-AI CR (OR 1.95) and increased pregnancy rate at 90 DIM (OR 2.50) and at 120 DIM (OR 1.77). Low milk production increased the probability of being detected in estrus at the end of synchronization within treated cows (OR 1.67), and interacted with treatment to increase probability of pregnancy at 90 DIM (OR 2.39) relative to control counterparts. It is suggested that when administered with efficient cooling, combined hormonal treatment in specific subgroups of cows, that is, those previously diagnosed with postpartum uterine disease or those with low body condition score or low milk yield might improve fertility during the summer and autumn. Integration of such an approach into reproductive management during the hot seasons might improve treatment efficiency and reduce expenses.

Effects of Escherichia coli- and Staphylococcus aureus-induced mastitis in lactating cows on oocyte developmental competence.

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.

Naturally occurring mastitis disrupts developmental competence of bovine oocytes.

We examined the effects of naturally occurring mastitis on bovine oocyte developmental competence in vitro. Specifically, we investigated the effects of intramammary infection on the ovarian pool of oocytes (i.e., follicle-enclosed oocytes) and their ability to undergo in vitro maturation, fertilization, and further development to the blastocyst stage. Culled Holstein cows (n=50) from 9 commercial dairy farms in Israel were allotted to 3 groups according to somatic cell count (SCC) records of the last 3 monthly milk tests as well as of quarter samples collected before slaughter: (1) low SCC (n=7), (2) medium SCC (n=16), or (3) high SCC (n=27). Means of SCC values differed among low-, medium-, and high-SCC groups: 148,000, 311,000 and 1,813,000 cell/mL milk, respectively. Milk yield and days in milk did not differ among the 3 groups. Bacterial isolates included coagulase-negative staphylococci, Escherichia coli, Streptococcus dysgalactiae, or no bacteria found. Ovaries were collected at the abattoir and brought to the laboratory. Cumulus oocyte complexes were recovered separately from each cow and subjected individually to in vitro maturation and fertilization, followed by 8d in culture. The number of aspirated oocytes did not differ among groups, with a range of 17 to 21 oocytes per cow. The proportion of oocytes that cleaved into 2- to 4-cell-stage embryos (86.1 ± 3.4%) did not differ among groups. In contrast, mean percentages of embryos developed to the blastocyst stage on d 7 and 8 after fertilization were less in both medium- and-high SCC groups than in the low-SCC group (5.6 ± 2.3 and 4.1 ± 1.8 vs. 18.1 ± 4.6%, respectively). Additional analysis indicated that cleavage and blastocyst-formation rates did not differ among the bacterial types in the low-, medium-, and high-SCC groups. These are the first results to demonstrate that naturally occurring mastitis disrupts the developmental competence of the ovarian pool of oocytes, (i.e., oocytes at the germinal vesicle stage). The disruption was associated with elevation of SCC rather than bacterial type. The results may provide a partial explanation for the low fertility of cows that have contracted mastitic pathogens before insemination.

Follicular characteristics and luteal development after follicle-stimulating hormone induced multiple ovulations in heifers.

A protocol based on small doses of FSH was examined for the induction of double or triple (multiple) ovulations in cattle. Ovulation rate, follicular characteristics, and luteal responses were determined. In Exp. 1, three groups of estrous-synchronized, cyclic Holstein heifers were treated once daily, on d 3 to 6 of the cycle, with a FSH product (Folltropin-V): large FSH dose (total of 150 mg; n=18), medium FSH dose (total of 130 mg, n=12), and small FSH dose (total of 80 mg; n=7). Controls received saline (n=6). Prostaglandin F(2α) was injected on d 6, ultrasound-guided aspiration of surplus follicles (if needed) was performed on d 7, and GnRH was injected on d 8 to induce ovulation. The large FSH dose induced growth of more (2.6±0.3, P<0.05) large follicles than controls on d 8; medium and small FSH doses insufficiently stimulated growth of <2 large follicles. Ovulation rates were determined in subgroups of heifers (n=10, 13, 4, and 6, respectively). The large FSH dose induced greater rates (P<0.01) of mostly double and triple ovulations (90% multiple ovulations, 70% double ovulations), most of which (89%) were bilateral, with only 2 out of 10 heifers requiring aspiration of surplus follicles. Medium and small FSH doses induced fewer multiple ovulations (38% and 25%, respectively). Estradiol concentrations on d 8 did not differ among treatments, but the concentration per large follicle in controls was greater (P<0.05) than in FSH treatments. Mean corpus luteum (CL) volume in single-ovulation controls was greater (P<0.05) than that of multiple ovulations in the large FSH group and total CL volume and progesterone concentrations were numerically greater in multiple ovulations. In Exp. 2, the characteristics of follicles aspirated on d 7 from large FSH (n=11) and control heifers (n=10) were compared. Based on estradiol-to-progesterone ratio, 57% of the large FSH-treated follicles were classified as codominant/healthy follicles and 43% as subordinate/early atretic. Although concentrations of estradiol and androstenedione in FSH-treated codominant follicles were less (P<0.05) than in controls, estradiol-to-progesterone ratio indicated that those follicles were steroidogenically active. Finely tuned small doses of FSH administered during the first follicular wave can induce a large incidence of double/triple, mainly bilateral, ovulations in cattle, which may serve as a basis for treatment aimed at promoting twinning in beef cattle.

Progesterone supplementation postinsemination improves fertility of cooled dairy cows during the summer.

Reduced fertility of dairy cows during periods of elevated temperature, humidity, or both might be associated with low plasma progesterone concentration. Alleviation of thermal stress by efficient cooling is a prerequisite for improving fertility by hormonal treatment. We examined whether insertion of a controlled intravaginal drug-releasing (CIDR) insert containing progesterone following artificial insemination (AI) would improve summer conception rate. Control (n = 195) and treated (CIDR; n=165) cows, yielding on average 42.3 kg milk/d, were inseminated following estrus detection during the summer (July to October) in 2 commercial dairy herds in Israel. Mean maximal air temperature and relative humidity during the study were 30.2°C and 86%, respectively. All experimental cows were efficiently cooled throughout the study, as confirmed by measuring the body temperature of random cows. Treated cows received a CIDR insert on d 5 ± 1 post-AI for 13 d and pregnancy was confirmed by palpation 45 d post-AI. Plasma progesterone concentration in treated cows was elevated by approximately 1.5 ng/mL. Multiple logistic regressions were used to analyze conception rate. Treatment did not alter the overall conception rate; however, probability of conception increased in CIDR-treated cows with low body condition score (BCS) compared with their control counterparts (53 vs. 27%, respectively). A pronounced increase in probability of conception was recorded in CIDR-treated cows exhibiting both low BCS and postpartum reproductive disorders, compared with their control counterparts (58 vs. 14%, respectively). Exogenous progesterone supplementation on d 5 post-AI for 13 d improves summer fertility of subpopulations of cows exhibiting low BCS and postpartum reproductive disorders. Reproductive management based on specific hormonal treatment of designated subgroups of cows known to derive beneficial effects from it might improve treatment efficiency and reduce expenses.

Association of conception rate with pattern and level of somatic cell count elevation relative to time of insemination in dairy cows.

The aim was to evaluate the effects of mastitis, determined by the pattern and level of somatic cell count (SCC) around first artificial insemination (AI), on conception rate (CR). Data from 287,192 first AI and milk records covering a 7-yr period were obtained from the Israeli Herd Book. Analyses examined the association of probability of conception with SCC elevation relative to timing of AI, using generalized linear mixed models. A SCC threshold of 150,000 cells/mL of milk was set to distinguish between uninfected cows and cows with mastitis. Accordingly, cows with high SCC before and low SCC after AI were designated cured, those with low SCC before and high SCC after AI were designated newly infected, and cows with high SCC before and after AI were designated chronic (likely subclinical) mastitic cows. Compared with uninfected cows, the cured, newly infected, and chronic subgroups showed reduced CR (39.4±0.1, 36.6±0.2, 32.9±0.3, and 31.5±0.2, respectively). In the chronic, subclinical group, probability of conception was lowered by 14.5% in the mild and moderately elevated SCC subgroups and by 20.5% in cows with high SCC elevation compared with the uninfected group (CR of 29.7 vs. 39.4%, respectively). A single high elevation of SCC (>10(6) cells/mL on only 1 milk test day) lowered the probability of conception by 23.6% when it occurred during the 10 d immediately before AI, but not when it occurred earlier. For 30 d after AI, probability of conception was lowered by about 23%, as reflected in a CR of about 27% compared with the uninfected group. Probability of conception was lowered in cows with uterine and foot health problems (33.9%), in multiparous cows (34.1%), and in cows in the summer (29.1%), but no interactions with mastitis were detected. Results indicate that SCC elevation around AI, typical for subclinical mastitis, was associated with a significant reduction in probability of conception, and that even mild SCC elevation reduced CR. Severe elevation of SCC before AI, typical for clinical intramammary infection, reduced the probability of conception.

Immediate and carryover effects of Gram-negative and Gram-positive toxin-induced mastitis on follicular function in dairy cows.

This study compared immediate and carryover effects of mastitis induced by Gram-negative endotoxin (E. coli LPS) and Gram-positive exosecretions (Staph. aureus ex.) on preovulatory follicle function. Synchronized, uninfected cyclic lactating Holstein cows were treated with PGF(2α) on day 6 of the cycle and 36 h later, a dose of either E. coli LPS (n = 8), S. aureus ex. (n = 10), or saline (n = 9) was administered into the mammary gland. Follicular fluids and granulosa cells were aspirated 6 h later from the preovulatory follicles and cows were treated with GnRH. This (cycle 1; immediate effect) was repeated three times (excluding the mammary injections) to induce three 7 d cycles (cycles 2, 3, and 4; carryover effect). E. coli LPS increased body temperature, plasma cortisol concentration, and somatic cell count (SCC), whereas S. aureus ex. induced a minor, subclinical elevation of SCC and slight rise (NS) in body temperature and cortisol concentration. Follicular estradiol, androstenedione, and progesterone concentrations in the E. coli LPS group decreased (P < 0.05) in cycle 1 to about 40%, 13%, and 35%, respectively, of control levels, whereas in the S. aureus ex. group, only estradiol decreased (P < 0.05), to 56% of control concentrations. In cycles 3 and 4, follicular steroids in the E. coli LPS group returned to control concentrations, whereas in the S. aureus ex. group, follicular concentrations of estradiol and androstenedione were lower (P < 0.10) than in controls. In the control group, the concentrations of all follicular and circulating steroids remained stable (P > 0.05) throughout the study. Follicle size was similar in all groups, but the S. aureus ex. treatment caused a decrease (P < 0.02) in the number of follicles developed in cycles 3 and 4. The mRNA expression of steroidogenic genes and LHCGR in the granulosa cells was not affected (P > 0.05) by either treatment during the study, except for a tendency toward lower (P < 0.1) expression in cycle 1 and lower (P < 0.05) expression in cycle 4 of the latter in the S. aureus ex. group. Strain levels, such as SCC and body temperature, following toxin injection correlated well with the magnitude of the immediate decline in follicular steroids. As is typical for Gram-negative clinical events, E. coli LPS-induced acute mastitis caused immediate, short-term, but not long-term impairment of follicular responses, whereas the Gram-positive S. aureus ex.-induced subclinical mastitis exhibited both immediate and carryover disruptive effects on preovulatory follicle function.

Subclinical, chronic intramammary infection lowers steroid concentrations and gene expression in bovine preovulatory follicles.

Chronic, subclinical intramammary infection depresses fertility. We previously found that 30% of subclinical mastitic cows exhibit delayed ovulation, low circulating estradiol levels, and delayed luteinizing hormone surge. We examined the function of preovulatory follicles of cows experiencing subclinical mastitis or a past event of acute clinical mastitis. Cows were diagnosed for mastitis by somatic cell count and bacteriological examination. All clinical infections were caused by Escherichia coli, and most subclinical infections were caused by Streptococcus dysgalactiae and coagulase-negative staphylococci. On day 6 of the cycle, cows received PGF2α; 42 h later, follicular fluids and granulosa cells or theca cells were aspirated from preovulatory follicles in vivo or following slaughter, respectively. Overall, follicular estradiol and androstenedione concentrations in the subclinical group (n = 28) were 40% lower (P < 0.05) than those in uninfected cows (n = 24) and lower than in past clinical mastitic cows (n = 9). Distribution analysis revealed a clear divergence among subclinical cows: one-third (9/28) exhibited low follicular estradiol; the other two-thirds had normal levels similar to all uninfected (P < 0.01) and most clinical cows (P < 0.08) that had normal follicular estradiol levels. Subclinical normal-estradiol cows had twofold higher (P < 0.05) circulating estradiol concentrations and sevenfold and fourfold higher (P < 0.05) follicular androstenedione levels and estradiol-to-progesterone ratio, respectively, than subclinical low-estradiol cows. Follicular progesterone level was not affected. Reduced expression (P < 0.05) of LHCGR in theca and granulosa cells, CYP11A1 (mRNA and protein) and CYP17A1 in theca cells, and CYP19A1 in granulosa cells may have contributed to the lower follicular steroid production in the subclinical low-estradiol subgroup. StAR and HSD3B1 in theca cells and FSHR in granulosa cells were not affected. Mastitis did not alter follicular growth dynamics, and no carryover effect of past clinical mastitis on follicular function was detected. These data indicate that a considerable proportion (one-third) of subclinical mastitic cows have abnormal follicular steroidogenesis, which can explain the reproductive failure associated with this disease.

Naturally occurring mastitis effects on timing of ovulation, steroid and gonadotrophic hormone concentrations, and follicular and luteal growth in cows.

The effects of naturally occurring subclinical chronic or clinical short-term mastitis on time of ovulation, plasma steroid and gonadotropin concentrations, and follicular and luteal dynamics were examined in 73 lactating Holstein cows. Cows were sorted by milk somatic cell count and bacteriological examination into an uninfected group (n=22), a clinical mastitis group (n=9; events occurring 20+/-7 d before the study), and a subclinical chronic mastitis group (n=42). In addition, uninfected and mastitic cows were further sorted by their estrus to ovulation (E-O) interval. About 30% of mastitic cows (mainly subclinical) manifested an extended E-O interval of 56+/-9.2h compared with 28+/-0.8h in uninfected cows and 29+/-0.5h in the other 70% of mastitic cows. In mastitic cows with extended E-O interval, the concentration of plasma estradiol at onset of estrus was lower than that of uninfected cows or mastitic cows that exhibited normal E-O intervals (3.1+/-0.4, 5.8+/-0.5, and 5.5+/-0.5 pg/mL, respectively). The disruptive effect of mastitis on follicular estradiol probably does not involve alterations in gonadotropin secretion because any depressive effects of mastitis on pulsatile LH concentrations were not detected. Cortisol concentrations did not differ among groups. The preovulatory LH surge in mastitic cows with delayed ovulation varied among individuals, being lower, delayed, or with no surge noted compared with the normal LH surge exhibited by uninfected cows or mastitic cows with normal E-O interval (6.8+/-0.7 ng/mL). The diameter of the second-wave dominant follicle was larger and the number of medium follicles was smaller in uninfected and subclinical cows with normal intervals compared with subclinical cows with extended intervals (13.4+/-0.5 vs. 10.9+/-0.9mm, and 3.8+/-0.2 vs. 6.7+/-0.14 follicles, respectively). Mid-luteal progesterone concentrations were similar in uninfected and mastitic cows. These results indicate for the first time that around 30% of cows with subclinical chronic mastitis exhibit delayed ovulation that is associated with low plasma concentrations of estradiol and a low or delayed preovulatory LH surge.

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.

Endocrine alterations associated with extended time interval between estrus and ovulation in high-yield dairy cows.

Short fertile half-lives of the male and female gametes in the female tract necessitate accurate timing of artificial insemination. We examined the possible association between extension of the estrus to ovulation (E-O) interval and alterations in concentrations of estradiol, progesterone, and the preovulatory LH surge before estrus and ovulation. High-yielding Holstein cows (n = 74 from a total of 106) were synchronized and were examined around the time of the subsequent estrus. They were observed continuously for estrual behavior. Blood samples were collected before and after estrus, and ultrasound checks for ovulation were made every 4 h. About three-quarters of the cows exhibited short (but normal) E-O intervals of 22 to 25 h (25%) or normal intervals of 25 to 30 h (47%); 17% of them displayed a long (but normal) E-O interval of 31 to 35 h, and about 10% exhibited a very long E-O interval of 35 to 50 h. Extended E-O interval comprised estrus-to-LH surge and LH surge-to-ovulation intervals that were both longer than normal. Pronounced changes in hormonal concentrations were noted before ovulation in the very long E-O interval group of cows: progesterone and estradiol concentrations were reduced, and the preovulatory LH peak surge was markedly less than in the other 3 groups. Postovulation progesterone concentrations during the midluteal phase were lesser in the very long and the long E-O interval groups compared with those in the short and normal interval groups. Season, parity, milk yield, and body condition did not affect the estrus to LH surge, LH surge to ovulation, and E-O intervals. The results indicate an association between preovulatory-reduced estradiol concentrations and a small preovulatory LH surge, on the one hand, and an extended E-O interval, on the other hand. Delayed ovulation could cause nonoptimal timing of AI, a less than normal preovulatory LH surge that may be associated with suboptimal maturation of the oocyte before ovulation, or reduced progesterone concentrations before and after ovulation. All may be factors associated with poor fertility in cows with a very long E-O interval.

Concentrações hormonais e desenvolvimento folicular de vacas leiteiras em hipertermia sazonal e aguda / Hormone concentration and follicular development in dairy cows under seasonal and acute hyperthermia

Avaliaram-se as concentrações hormonais e os parâmetros de desenvolvimento folicular de vacas leiteiras expostas ao calor sazonal e agudo. Dividiram-se os animais em quatro grupos: verão (n=5), outono (n=5), inverno com hipertermia aguda (grupo câmara climática, (CC), n=5) e inverno (n=9). Os animais foram abatidos no sétimo dia após a ovulação, e os parâmetros de desenvolvimento folicular avaliados. O líquido folicular do maior folículo foi aspirado e armazenado para posterior análise de hormônios esteróides e inibina. O número de células da granulosa vivas no verão e no outono foi 40 e 45 por cento respectivamente, menor que no inverno (P<0,05). A concentração de estradiol (E2) no inverno foi 62 por cento maior que no outono (P<0,05) e 34 por cento superior ao grupo verão (P<0,06). Houve um aumento na quantidade de androstenediona no verão em relação aos grupos inverno (P<0,08) e outono (P<0,05). A concentração de inibina foi maior no inverno do que no verão e CC (P<0,05). A exposição ao calor sazonal e agudo modificou os parâmetros de desenvolvimento do folículo e as concentrações hormonais no líquido folicular, podendo explicar em parte a queda nas taxas de concepção no verão.

The present study evaluated the seasonal and acute heat stress on follicular development and steroid and inhibin concentrations in follicular fluid, in bovine dominant follicle. Cows were distributed into four treatments: summer (n=5), autumn (n=5), animals heat stressed during the winter (n=5) and winter (n=9). On day 7 of the estrous cycle, animals were slaughtered and parameters related to follicle development were evaluated. The follicular fluid (FF) was aspirated and stored for further hormonal analysis. During the summer, the number of viable granulosa cells was 40 percent lower than during the winter, and there was a 45 percent decrease in this parameter during the autumn (P<0.05). In the winter, estradiol concentration was 62 percent higher than during the autumn (P<0.05) and 42 percent higher than during the summer (P<0.06). There was an increase in androstenedione concentration in summer group, when compared to winter (P<0.08) and autumn (P<0.05) groups. Inhibin concentration was higher in winter groups than summer and winter heat stressed groups (P<0.05). Seasonal and acute heat stress altered developmental parameters in dominant follicle and hormonal concentration in follicular fluid, those effects can partially explain the decrease in conception rates during summer.

Effects of GnRH administered to cows at the onset of estrus on timing of ovulation, endocrine responses, and conception.

Two experiments examined effects of GnRH administered within 3 h after onset of estrus (OE) on ovulation and conception in dairy cows. In experiment 1, 46 cows received either saline, 250 microg of GnRH, or 10 microg of the GnRH analogue, Buserelin. Cows were observed for estrus, blood samples were collected, and ovulations were monitored by ultrasound. In controls, 76% of cows had intervals from estrus to ovulation of < or = 30 h and 24% had intervals > 30 h. Treatment with either GnRH or GnRH analogue (data combined) increased magnitude of LH surges and decreased intervals from estrus to LH surge or to ovulation. Treated cows all ovulated < or = 30 h after OE. Among control cows, plasma estradiol concentrations before estrus correlated positively with amplitudes of LH surges. Higher plasma progesterone was observed in the subsequent estrous cycle in GnRH-treated cows compared to control cows with delayed ovulations. Experiment 2 included 152 primiparous and 211 multiparous cows in summer and winter. Injection of GnRH analogue at OE increased conception rates (CR) from 41.3 to 55.5% across seasons. In summer, GnRH treatment increased CR from 35.1 to 51.6%. Across seasons, GnRH increased CR from 36.0 to 61.5% in cows with lower body condition at insemination and GnRH increased CR (63.2 vs. 42.2%) in primiparous cows compared to controls. Use of GnRH eliminated differences in CR for cows inseminated early or late relative to OE and increased CR in cows having postpartum reproductive disorders. In conclusion, GnRH at onset of estrus increased LH surges, prevented delayed ovulation, and may increase subsequent progesterone concentrations. Treatments with GnRH increased conception in primiparous cows, during summer, and in cows with lower body condition.

Erythropoiesis regulation during the development of ascites syndrome in broiler chickens: a possible role of corticosterone.

The ascites syndrome in broiler chickens is attributed to metabolic burdening, which results from intensive genetic selection for rapid growth coupled with exposure to extreme environmental conditions, such as low ambient temperature. These conditions impose on the broilers difficulties in fulfilling tissue demands for oxygen, and the birds exhibit a decrease in blood oxygen saturation and high hematocrit values. It is unknown whether the increase in hematocrit results from alteration in erythropoiesis or from fluid exudation out of the blood system to the abdominal cavity. The present study was conducted to examine the association between abnormal stress response and erythropoiesis process in ascitic broilers. Ascitic chickens revealed a uniquely continuous stress response: expressing an increase (P < or = 0.05) in plasma corticosterone concentration 2 to 3 wk before death. At 5 wk of age, ascitic broilers exhibited an increase (P < 0.05) in hematocrit, blood cell count, and packed cells and blood volumes, with no significant change in plasma volume. These results confirm an accelerated erythropoiesis process in ascitic birds. Increased blood cell production in ascitic birds was matched by an increase (P < 0.05) in the proportion of immature red blood cells (23%) in comparison with broilers that remained healthy (7%), and by decreased (P < 0.05) hemoglobin content relative to red blood cells. We conclude that continually increased corticosterone concentrations, as an inducer of erythropoiesis proliferation and differentiation arrest, in ascitic chickens, resulted in increased production of red blood cells (partially immature) with decreased hemoglobin content; this decrease in hemoglobin might have contributed to enhanced development of hypoxemia and to aggravation of the syndrome.

Effect of treatment with follicle-stimulating hormone or bovine somatotropin on the quality of oocytes aspirated in the autumn from previously heat-stressed cows.

Conception in dairy cows during autumn remains low even after summer temperatures decline. This is possibly a residual effect of heat stress on oocyte quality. Lactating Holsteins previously heat-stressed during summer were used in two experiments (n = 8 and 16 cows) in autumn to examine hormonal strategies for improving quality of oocytes. Follicles (3 to 8 mm in diameter) were aspirated by the ovum pick-up procedure on d 4 of two consecutive estrous cycles of treated and control cows. Oocytes were classified morphologically, matured in vitro, chemically activated, and cultured for 8 d. In Experiment 1, FSH (2 x 200 mg, at a 12-h interval) was injected on d 5 and 12 of cycle 1 (treated cycle). Before FSH, the percentage of good quality oocytes (grade I) and the cleavage rate averaged 52 and 24%, respectively, in both groups. During the FSH-treated cycle, more 6- to 9-mm follicles were observed. In the subsequent cycle, rates of grade I oocytes and cleavage were significantly higher in FSH-treated than in control cows (89 vs. 51% and 85 vs. 31%, respectively). In Experiment 2, recombinant bovine somatotropin (bST, 500 mg) was injected on d 4 and 18 of the treated cycle. The bST treatment increased the number of 3- to 5-mm follicles. Before bST, grade I (39%) and cleaved oocytes (40%), were similar across treatment groups. In the subsequent cycle, the percentage of grade I oocytes was significantly higher in treated than in control cows (72 vs. 26%), but cleavage rates were similar. Neither FSH or bST improved blastocyst formation, and regardless of treatment, few blastocysts were formed. Treatment with bST improved oocyte morphology, whereas FSH improved both oocyte morphology and cleavage rates during autumn following summer heat stress.

Seasonal differences in progesterone production by luteinized bovine thecal and granulosa cells.

This study examined seasonal differences in progesterone (P4) production by granulosa cells (GC) and thecal cells (TC) that were luteinized in vitro during the winter or the summer; it also compared plasma P4 concentrations of lactating dairy cows in the two seasons. First-wave dominant follicles obtained from Holstein cows were dissected on day 6 of the cycle, GC and TC were separated, enzymatically dispersed, and cultured for 9 days in media containing 1% fetal calf serum, forskolin (10 micromol/mL) and insulin (2 microg/mL), to induce cell luteinization. All experimental procedures were identical and characteristics of the follicles were similar in the two seasons. During 9 days of culture, P4 production by luteinized GC was higher in winter than in summer, but the difference only tended to be significant. In contrast, luteinized TC produced three times as much P4 in winter as in summer (324 versus 100 ng/10(5)cells). In the in vivo experiment, P4 concentrations in plasma collected during entire estrous cycles in winter and summer were compared. The cows were, on average, at 70 days postpartum and yielded similar amounts of milk. Concentrations of progesterone in plasma were significantly higher in winter than in summer; during the mid-luteal phase the difference between the two seasons was 1.5 ng/mL. These results indicate that chronic effects of heat-stress are possibly carried over from an impaired follicle to an impaired corpus luteum (CL), and that luteinized TC are more susceptible to heat-stress than luteinized GC.