Effects of intravenous infusion of E. coli lipopolysaccharide in early pregnant cows.

The objective was to characterize effects of Escherichia coli LPS (given i.v.) on corpus luteum (CL) and embryonic viability in early pregnant cattle. Eight non-lactating German Holstein cows were given 0.5 µg/kg LPS on 35 ± 3 day (mean ± s.e.m.) of pregnancy, whereas seven heifers, 41 ± 6 day pregnant, were given 10 mL saline (control group). Transrectal B-mode examinations of the CL were done at -1, 3, 6, 12, 24, 48, 72 and 96 h relative to treatment. Blood samples were collected at -1, 0.5, 1, 2, 3, 4, 6, 9, 12, 24, 48, 72 and 96 h. At 12 and 48 h, the CL was biopsied. None of the cows still in the experiment 10 day after LPS (n = 7) had embryonic loss. In LPS-treated cows, luteal area decreased (from 4.1 to 3.1 cm2; P ≤ 0.05) within 6 h and until 48 h. Luteal blood flow decreased by 39% (P ≤ 0.05) within the first 6 h after LPS, but returned to pre-treatment values by 48 h. Plasma P4 decreased by 62% (P ≤ 0.05), reached a nadir (2.7 ± 0.6 ng/mL) at 12 h after LPS and was not restored to pre-treatment (P ≤ 0.05). In luteal tissue, mRNAs for STAR and for FGF1 were lower (P ≤ 0.05) in LPS than in saline-treated cattle at 12 h, with no difference between groups at 48 h. Levels of mRNAs for CASP3 and FGF2 were not different between groups (P > 0.05) at 12 or 48 h after treatment. In conclusion, LPS transiently suppressed CL function, but did not induce embryonic mortality.

Testicular hyperthermia increases blood flow that maintains aerobic metabolism in rams.

There is a paradigm that testicular hyperthermia fails to increase testicular blood flow and that an ensuing hypoxia impairs spermatogenesis. However, in our previous studies, decreases in normal and motile spermatozoa after testicular warming were neither prevented by concurrent hyperoxia nor replicated by hypoxia. The objective of the present study was to determine the effects of increasing testicular temperature on testicular blood flow and O2 delivery and uptake and to detect evidence of anaerobic metabolism. Under general anaesthesia, the testicular temperature of nine crossbred rams was sequentially maintained at ~33°C, 37°C and 40°C (±0.5°C; 45min per temperature). As testicular temperature increased from 33°C to 40°C there were increases in testicular blood flow (13.2±2.7 vs 17.7±3.2mLmin-1 per 100g of testes, mean±s.e.m.; P<0.05), O2 extraction (31.2±5.0 vs 47.3±3.1%; P<0.0001) and O2 consumption (0.35±0.04 vs 0.64±0.06mLmin-1 per 100g of testes; P<0.0001). There was no evidence of anaerobic metabolism, based on a lack of change in lactate, pH, HCO3- and base excess. In conclusion, these data challenge the paradigm regarding scrotal-testicular thermoregulation, as acute testicular hyperthermia increased blood flow and tended to increase O2 delivery and uptake, with no indication of hypoxia or anaerobic metabolism.

Role of the Na+/K+-ATPase ion pump in male reproduction and embryo development.

Na+/K+-ATPase was one of the first ion pumps studied because of its importance in maintaining osmotic and ionic balances between intracellular and extracellular environments, through the exchange of three Na+ ions out and two K+ ions into a cell. This enzyme, which comprises two main subunits (α and ß), with or without an auxiliary polypeptide (γ), can have specific biochemical properties depending on the expression of associated isoforms (α1ß1 and/or α2ß1) in the cell. In addition to the importance of Na+/K+-ATPase in ensuring the function of many tissues (e.g. brain, heart and kidney), in the reproductive tract this protein is essential for embryo development because of its roles in blastocoel formation and embryo hatching. In the context of male reproduction, the discovery of a very specific subunit (α4), apparently restricted to male germ cells, only expressed after puberty and able to influence sperm function (e.g. motility and capacitation), opened a remarkable field for further investigations regarding sperm biology. Therefore, the present review focuses on the importance of Na+/K+-ATPase on male reproduction and embryo development.

Dairy farmers' perceptions toward the implementation of on-farm Johne's disease prevention and control strategies.

Implementation of specific management strategies on dairy farms is currently the most effective way to reduce the prevalence of Johne's disease (JD), an infectious chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). However, dairy farmers often fail to implement recommended strategies. The objective of this study was to assess perceptions of farmers participating in a JD prevention and control program toward recommended practices, and explore factors that influence whether or not a farmer adopts risk-reducing measures for MAP transmission. Semi-structured interviews were conducted with 25 dairy farmers enrolled in a voluntary JD control program in Alberta, Canada. Principles of classical grounded theory were used for participant selection, interviewing, and data analysis. Additionally, demographic data and MAP infection status were collected and analyzed using quantitative questionnaires and the JD control program database. Farmers' perceptions were distinguished according to 2 main categories: first, their belief in the importance of JD, and second, their belief in recommended JD prevention and control strategies. Based on these categories, farmers were classified into 4 groups: proactivists, disillusionists, deniers, and unconcerned. The first 2 groups believed in the importance of JD, and proactivists and unconcerned believed in proposed JD prevention and control measures. Groups that regarded JD as important had better knowledge about best strategies to reduce MAP transmission and had more JD risk assessments conducted on their farm. Although not quantified, it also appeared that these groups had more JD prevention and control practices in place. However, often JD was not perceived as a problem in the herd and generally farmers did not regard JD control as a "hot topic" in communications with their herd veterinarian and other farmers. Recommendations regarding how to communicate with farmers and motivate various groups of farmers according to their specific perceptions were provided to optimize adoption of JD prevention and control measures and thereby increase success of voluntary JD control programs.

Pregnancy Loss in Dairy Cattle: Relationship of Ultrasound, Blood Pregnancy-Specific Protein B, Progesterone and Production Variables.

Objectives were to determine associations between percentage pregnancy loss (PPL) in dairy cattle and: (i) pregnancy diagnosis by ultrasonography; (ii) pregnancy diagnosis by serum pregnancy-specific protein B (PSPB) concentrations, with or without serum progesterone concentrations; and (iii) production and environmental factors. This study included 149 822 pregnancy diagnoses conducted over 13 years in Holstein-Friesian cows in Hungarian dairy herds. The following were determined: PPL in cows diagnosed pregnant by transrectal ultrasonography 29-42 days after artificial insemination (AI; n = 11 457); PPL in cows diagnosed pregnant by serum PSPB 29-35 days after AI (n = 138 365); and PPL and its association with serum progesterone concentrations, PSPB and production/environmental variables. The definition of PPL was percentage of cows initially diagnosed pregnant based on ultrasonography or PSPB, but not pregnant when examined by transrectal palpation 60 -70 days after AI. The PPL was lower (p < 0.001) in cows following ultrasonographic vs PSPB diagnosis of pregnancy at 29-35 days (8.1 vs 19.3%, respectively), but was higher in cows following ultrasonographic pregnancy diagnosis on 29-35 vs 36-42 days (8.1 vs 7.1%, respectively, P < 0.05). Furthermore, 72.9% of pregnancies with ultrasound-detected morphological abnormalities resulted in pregnancy loss. As a subset of PSPB data, a fully quantitative PSPB assay was used for 20 430 samples; PPL in cows with a high PSPB concentration (>1.1 ng/ml) was lowest (15.0%), whereas cows with low concentrations of both PSPB and progesterone (0.6-1.1 and <2 ng/ml, respectively) had the highest PPL (76.3%; p < 0.0001). Furthermore, PPL was higher in cows with advanced parity and with high milk production, when ambient temperatures were high, although body condition score (BCS) had no effect on PPL. Finally, there were no significant associations between serum PSPB and environmental temperatures or number of post-partum uterine treatments.

Invited review: Changes in the dairy industry affecting dairy cattle health and welfare.

The dairy industry in the developed world has undergone profound changes over recent decades. In this paper, we present an overview of some of the most important recent changes in the dairy industry that affect health and welfare of dairy cows, as well as the science associated with these changes. Additionally, knowledge gaps are identified where research is needed to guide the dairy industry through changes that are occurring now or that we expect will occur in the future. The number of farms has decreased considerably, whereas herd size has increased. As a result, an increasing number of dairy farms depend on hired (nonfamily) labor. Regular professional communication and establishment of farm-specific protocols are essential to minimize human errors and ensure consistency of practices. Average milk production per cow has increased, partly because of improvements in nutrition and management but also because of genetic selection for milk production. Adoption of new technologies (e.g., automated calf feeders, cow activity monitors, and automated milking systems) is accelerating. However, utilization of the data and action lists that these systems generate for health and welfare of livestock is still largely unrealized, and more training of dairy farmers, their employees, and their advisors is necessary. Concurrently, to remain competitive and to preserve their social license to operate, farmers are increasingly required to adopt increased standards for food safety and biosecurity, become less reliant on the use of antimicrobials and hormones, and provide assurances regarding animal welfare. Partly because of increasing herd size but also in response to animal welfare regulations in some countries, the proportion of dairy herds housed in tiestalls has decreased considerably. Although in some countries access to pasture is regulated, in countries that traditionally practiced seasonal grazing, fewer farmers let their dairy cows graze in the summer. The proportion of organic dairy farms has increased globally and, given the pressure to decrease the use of antimicrobials and hormones, conventional farms may be able to learn from well-managed organic farms. The possibilities of using milk for disease diagnostics and monitoring are considerable, and dairy herd improvement associations will continue to expand the number of tests offered to diagnose diseases and pregnancy. Genetic and genomic selection for increased resistance to disease offers substantial potential but requires collection of additional phenotypic data. There is every expectation that changes in the dairy industry will be further accentuated and additional novel technologies and different management practices will be adopted in the future.

Calm temperament improves reproductive performance of beef cows.

Profitability of a beef operation is determined by the proportion of cows attaining pregnancy early in the breeding season and those that are pregnant at the end of breeding season. Many factors, including temperament, contribute to those reproductive parameters. The objective of this study was to evaluate effects of temperament on reproductive performance of beef cows. In Experiment 1, Angus and Angus-cross beef cows (n = 1546) from eight locations were assigned a body condition score (BCS; 1 = emaciated; 9 = obese) and chute exit and gait score (1 = slow exit, walk; calm temperament; 2 = jump, trot or run; excitable temperament). Cows were grouped with bulls (1 : 25 to 1 : 30; with satisfactory breeding potential and free of venereal disease) for an 85-day breeding season. Pregnancy status and stage of gestation were determined (transrectal palpation) 35 days after the end of the breeding season. Controlling for BCS (p < 0.01) and handling facility (p < 0.0001) and handling facility by temperament score interaction (p < 0.001), breeding season pregnancy rate was lower in excited versus calm cows [88.6% (798/901) vs 94.1% (607/645); p < 0.001]. Cows with an excitable temperament took 24 more days to become pregnant compared to calm cows (median days to pregnancy, 35 vs 59 days; p < 0.0001). In Experiment 2, Angus and Angus-cross beef cows (n = 1407) from 8 locations were assigned scores for body condition and chute exit and gait (as described in Experiment 1) and assigned to bulls (breeding sound and free of venereal disease; 1 : 25 to 1 : 30) for 85 days. Pregnancy status was determined by transrectal palpation at 2 and 6 months after the onset of the breeding season. Controlling for BCS (p < 0.05), pregnancy loss was higher in excited versus calm cows [5.5% (36/651) vs 3.2% (20/623), p < 0.0001]. In conclusion, beef cows with an excitable temperament had significantly lower reproductive performance than calmer cows. The modified two-point chute exit-gait scoring method was repeatable and identified cattle with an excitable temperament.

Serial frequent or multiple Tru-cut® testicular biopsies in rams enable assessment of histological characteristics or transcriptional profiles, with no acute or chronic adverse effects.

This study aimed to evaluate the feasibility of performing multiple testicular biopsies in rams using Tru-cut® needles, assessing histological structure, gene expression, and potential complications such as effects on semen quality, testicular blood flow, and ultrasonographic echotexture. In Exp. 1, six mature rams underwent testicular biopsies at intervals (0, 3, 6, 12, 24, and 48 h) using a 16 G Tru-cut® needle, with alternating testes for each collection. Benzathine benzylpenicillin and flunixin meglumine were administered for infection and inflammation control. Local anesthesia and post-biopsy care included lidocaine, digital pressure, and ice application. Testicular samples were analyzed for gene expression related to inflammation, oxidative stress, and steroidogenesis. Semen quality was assessed pre-biopsy and 28 days post-biopsy. Ultrasonographic evaluations of the scrotum and testes were conducted before biopsies and on days 5, 9, 13, 17, and 21 post-biopsies. In Exp. 2, a second group of six mature rams underwent biopsies using 14 G needles, with two samples taken from each testis. Samples were histologically examined for structural preservation. Scrotal skin temperature was measured using infrared thermography, and testicular blood flow was assessed via color Doppler ultrasonography, with measurements taken before and on days 1, 2, 4, 6, 8, 10, 25, 50, 75, and 100 post-biopsies. Semen collection followed the same schedule as in Exp. 1. In Exp. 3, biopsies were performed on different testicular regions (upper, middle, lower) using 12 G, 14 G, and 16 G needles to compare structural preservation. Samples were histologically analyzed. No clinical signs of injury, inflammation, or fluid accumulation were observed. Scrotal pain, increased temperature, swelling, and bleeding were absent, and behavioral signs indicative of pain were not detected. Gene expression remained unchanged, and no significant alterations in seminal characteristics or testicular echogenicity were observed. A slight increase in resistivity and pulsatility indices was noted in Exp. 2. Biopsies with 14 G and 16 G needles resulted in structural disruptions, while 12 G needles better preserved testicular parenchyma. Multiple testicular biopsies using Tru-cut® needles did not cause significant morphological changes, alter transcriptional profiles, or affect semen or ultrasonographic characteristics, demonstrating that this method is viable for monitoring acute molecular changes in the testes.

Escherichia coli lipopolysaccharide administration transiently suppresses luteal structure and function in diestrous cows.

The objective was to characterize the effects of Escherichia coli lipopolysaccharide (LPS) endotoxin (given i.v.) on luteal structure and function. Seven nonlactating German Holstein cows, 5.1 ± 0.8 years old (mean ± s.e.m.), were given 10  ml saline on day 10 (ovulation=day 1) of a control estrous cycle. On day 10 of a subsequent cycle, they were given 0.5 µg/kg LPS. Luteal size decreased (from 5.2 to 3.8 cm², P≤0.05) within 24 h after LPS treatment and remained smaller throughout the remainder of the cycle. Luteal blood flow decreased by 34% (P≤0.05) within 3 h after LPS and remained lower for 72 h. Plasma progesterone (P4) concentrations increased (P≤0.05) within the first 3 h after LPS but subsequently declined. Following LPS treatment, plasma prostaglandin (PG) F metabolites concentrations were approximately tenfold higher in LPS-treated compared with control cows (9.2 vs 0.8 ng/ml, P≤0.05) within 30 min, whereas plasma PGE concentrations were nearly double (P≤0.05) at 1 h after LPS. At 12 h after treatment, levels of mRNA encoding Caspase-3 in biopsies of the corpus luteum (CL) were increased (P≤0.05), whereas those encoding StAR were decreased (P≤0.05) in cattle given LPS vs saline. The CASP3 protein was localized in the cytoplasm and/or nuclei of luteal cells, whereas StAR was detected in the cytosol of luteal cells. In the estrous cycle following treatment with either saline or LPS, there were no significant differences between groups on luteal size, plasma P4 concentrations, or gene expression. In conclusion, LPS treatment of diestrus cows transiently suppressed both the structure and function of the CL.

Short communication: Prepartum plasma insulin-like growth factor-I concentrations based on day of insemination are lower in cows developing postpartum diseases.

Because peripartal production diseases are prevalent in dairy cows, early recognition is crucial. Several studies reported metabolic variables as risk predictors for subsequent diseases. To improve on-farm testing and application of those methods, the sampling procedure should take into account variation in gestation length. Furthermore, additional variables indicating cows at risk of any production disease should be sought. Therefore, the objective was to characterize differences between cows with and without postpartum production disease (retained fetal membranes, ketosis, hypocalcemia, abomasal displacement, metritis, mastitis) by prepartum measurement of serum nonesterified fatty acid (NEFA) and plasma insulin-like growth factor (IGF)-I concentrations relative to the artificial insemination (AI) that established pregnancy. Blood was collected from 41 Holstein Friesian cows on 235 to 241, 242 to 248, 249 to 255, 256 to 262, 263 to 269, 270 to 276, 277 to 283, and 284 to 290 d after AI. Health status was assessed daily for 3 wk after calving; 25 cows (66%) had at least one production disease. Cows developing postpartum diseases had higher mean serum NEFA concentrations (450 ± 26 µmol/L; mean ± SE) and lower plasma IGF-I concentrations (78 ± 6 ng/mL) prepartum compared with healthy cows (259 ± 19 µmol/L and 117 ± 8 ng/mL, respectively). In conclusion, because of substantial variation among cows in gestation length, blood samples should be collected and studies performed on risk prediction relative to AI rather than expected date of calving. As the somatotropic axis is one of the key regulators of metabolic adaption for onset of lactation, IGF-I might be a useful variable to differentiate between cows susceptible to production diseases and cows that are able to adapt adequately within the transition period and remain healthy.

Ultrasonography for monitoring reproductive function in the bull.

Diagnostic ultrasonography has been widely used for examination of the reproductive tract of female cattle, but more sparingly in bulls. Typical clinical ultrasonographic examinations of bull testes are unlikely to affect semen quality or sperm production. The ultrasonographic anatomy of bull testes and accessory sex glands has been reported. Although testicular echogenicity increased (i.e. the parenchyma appeared more white) as a bull approached puberty, echogenicity was not superior to scrotal circumference as a predictor of puberty. Ultrasonography can be used to detect and characterize testicular pathology. It is noteworthy that areas of increased echogenicity (testicular fibrosis) are common, especially in young bulls, but are not associated with decreased semen quality (e.g. percentage of morphologically abnormal sperm). Neither visual evaluation nor computerized pixel analysis of testicular ultrasonic echotexture was consistently predictive of semen quality in bulls. Therefore, we concluded that the primary clinical use of ultrasonography in assessment of reproductive function in the bull is characterization of grossly detectable lesions in the testes and scrotum.

The effects of increased testicular temperature on testis-specific isoform of Na+/K+ -ATPase in sperm and its role in spermatogenesis and sperm function.

Impaired testicular thermoregulation is commonly implicated in abnormal spermatogenesis and impaired sperm function in animals and humans, with outcomes ranging from subclinical infertility to sterility. Bovine testes must be maintained 4-5 °C below body-core temperature for normal spermatogenesis. The effects of elevated testicular temperature have been extensively studied in cattle using a scrotal insulation model, which results in abnormal spermatogenesis and impaired sperm morphology and function. Using this model and proteomic approaches, we compared normal and abnormal sperm (from the same bulls) to elucidate the molecular basis of impaired function. We identified a cohort of sperm functional proteins differentially expressed between normal vs abnormal sperm, including a testis-specific isoform of Na(+) /K(+) -ATPase. In addition to its role as a sodium pump regulating sperm motility, Na(+) /K(+) -ATPase is also involved as a signalling molecule during sperm capacitation. In conclusion, because of its involvement in regulation of sperm function, this protein has potential as a fertility marker. Furthermore, comparing normal vs abnormal sperm (induced by scrotal insulation) is a useful model for identifying proteins regulating sperm function.

A new paradigm regarding testicular thermoregulation in ruminants?

Increased testicular temperature reduces percentages of morphologically normal and motile sperm and fertility. Specific sperm defects appear at consistent intervals after testicular hyperthermia, with degree and duration of changes related to intensity and duration of the thermal insult. Regarding pathogenesis of testicular hyperthermia on sperm quality and fertility, there is a long-standing paradigm that: 1) testes operate near hypoxia; 2) blood flow to the testes does not increase in response to increased testicular temperature; and 3) an ensuing hypoxia is the underlying cause of heat-induced changes in sperm morphology and function. There are very limited experimental data to support this paradigm, but we have data that refute it. In 2 × 3 factorial studies, mice and rams were exposed to two testicular temperatures (normal and increased) and three concentrations of O2 in inspired air (hyperoxia, normoxia and hypoxia). As expected, increased testicular temperature had deleterious effects on sperm motility and morphology; however, hyperoxia did not prevent these changes nor did hypoxia replicate them. In two follow-up experiments, anesthetized rams were sequentially exposed to: 1) three O2 concentrations (100, 21 and 13% O2); or 2) three testicular temperatures (33, 37 and 40 °C). As O2, decreased, testis maintained O2 delivery and uptake by increasing testicular blood flow and O2 extraction, with no indication of anaerobic metabolism. Furthermore, as testicular temperature increased, testicular metabolic rate nearly doubled, but increased blood flow and O2 extraction prevented testicular hypoxia and anaerobic metabolism. In conclusion, our data, in combination with other reports, challenged the paradigm that testicular hyperthermia fails to increase testicular blood flow and the ensuing hypoxia disrupts spermatogenesis.

Amelioration of heat stress-induced damage to testes and sperm quality.

Heat stress (HS) occurs when temperatures exceed a physiological range, overwhelming compensatory mechanisms. Most mammalian testes are ∼4-5 °C cooler than core body temperature. Systemic HS or localized warming of the testes affects all types of testicular cells, although germ cells are more sensitive than either Sertoli or Leydig cells. Increased testicular temperature has deleterious effects on sperm motility, morphology and fertility, with effects related to extent and duration of the increase. The major consequence of HS on testis is destruction of germ cells by apoptosis, with pachytene spermatocytes, spermatids and epididymal sperm being the most susceptible. In addition to the involvement of various transcription factors, HS triggers production of reactive oxygen species (ROS), which cause apoptosis of germ cells and DNA damage. Effects of HS on testes can be placed in three categories: testicular cells, sperm quality, and ability of sperm to fertilize oocytes and support development. Various substances have been given to animals, or added to semen, in attempts to ameliorate heat stress-induced damage to testes and sperm. They have been divided into various groups according to their composition or activity, as follows: amino acids, antibiotics, antioxidant cocktails, enzyme inhibitors, hormones, minerals, naturally produced substances, phenolic compounds, traditional herbal medicines, and vitamins. Herein, we summarized those substances according to their actions to mitigate HS' three main mechanisms: oxidative stress, germ cell apoptosis, and sperm quality deterioration and testicular damage. The most promising approaches are to use substances that overcome these mechanisms, namely reducing testicular oxidative stress, reducing or preventing apoptosis and promoting recovery of testicular tissue and restoring sperm quality. Although some of these products have considerable promise, further studies are needed to clarify their ability to preserve or restore fertility following HS; these may include more advanced sperm analysis techniques, e.g. sperm epigenome or proteome, or direct assessment of fertilization and development, including in vitro fertilization or breeding data (either natural service or artificial insemination).

Metabolic biomarkers, body condition, uterine inflammation and response to superovulation in lactating Holstein cows.

The objective was to determine associations between response to superovulation and body condition, subclinical endometritis and circulating metabolic biomarkers [adiponectin, leptin, insulin, IGF1, tumor necrosis factor (TNF) α, interleukin (IL) 1ß, IL6, and urea] in lactating dairy cows. Ten multiparous lactating Holstein cows in each body condition score (1-5; 1 emaciated; 5 obese) category (BCSC) 2.00 to < 2.50 (BCSC1), 2.50 to < 3.00 (BCSC2), 3.00 to <3.50 (BCSC3), 3.50 to <4.00 (BCSC4) and 4.00 to 5.00 (BCSC5) groups (total n = 50) were randomly selected and superovulated, timed artificially inseminated with frozen-thawed semen from three sires and embryos collected (n = 50 collections). At embryo collection, blood samples and embryo recovery fluid were collected for determination of metabolic markers and presence of subclinical endometritis (lavage technique; > 6% PMN). In total, 379 embryos were collected (average of 7.6 embryos per superovulation). Mean numbers of total ova and embryos was greater for cows in BCSC2, BCSC3 and BCSC4 groups compared with cows in BCSC1 and BCSC5 groups (P < 0.01). Total number of transferrable embryos were greater for cows in BCSC 2 and BCSC3 groups compared with cows in BCSC1, BCSC4 and BCSC5 groups (P < 0.01). Mean number of total ova and embryos and of transferrable embryos was higher for cows with 0 or 1-6% PMN compared to cows with >6% PMN (P < 0.01). In addition, there was a quadratic association between blood urea nitrogen concentrations and % transferrable embryos (r2 = 0.85; P < 0.05) and between BCS and % transferrable embryos (r2 = 0.73; P < 0.05). Circulating adiponectin, leptin, insulin, IGF1 and TNFα were greater in cows with moderate to good body condition compared to thin or obese cows (P < 0.05). Circulating adiponectin, leptin, IGF1 and insulin were greater in normal cows (≤6% PMNs), whereas, TNFα and IL1ß and IL6 were greater in cows with subclinical endometritis (P < 0.05). In conclusion, BCS and subclinical endometrial inflammation were associated with superovulatory response and embryo quality. Further, circulating metabolic biomarkers were associated with superovulatory response and embryo quality, likely due to donor's metabolic status and uterine environment. Optimizing superovulatory responses and embryo quality in lactating dairy cows requires management of nutrition and uterine health.

Acute mild heat stress alters gene expression in testes and reduces sperm quality in mice.

Heat stress is a major concern in animal reproduction, as testicular temperature must be 3-5 °C below body core temperature for production of motile and fertile sperm in mammals. Although recent studies concluded that increased temperature per se was the underlying pathophysiology of testicular impairment, more studies are required to better understand the mechanisms. Therefore, our objective was to investigate the impacts of mild acute heat stress on sperm and testes, and based on mRNA, elucidate involvement of StAR, Trp53 and Trp53-dependent intrinsic and extrinsic apoptotic pathways in pathophysiology of testicular heat stress. Forty-eight C57 BCL6 elite male mice were equally allocated into six groups, anesthetized and the distal third of their body immersed in a water-bath at 40 or 30 °C (heat treatment and control, respectively) for 20 min. Intervals from heat exposure (Day 0) to euthanasia were: 8 and 24 h and 7, 14 and 21 d (plus a control group at 14 d). The epididymides were excised, minced and placed in Tyrode albumin lactate pyruvate hepes (TALPH) at 37 °C for 15 min to recover sperm. Based on computer assisted sperm analysis (CASA), heat treatment reduced total and progressive motility ∼40% (P < 0.05) on Days 14 and 21. Furthermore, percentage morphologically normal sperm was significantly decreased on Day 7, with greater reductions on Days 14 and 21, mostly due to increased midpiece defects. Acrosome integrity (FITC PSA) was decreased ∼35% at 8 h (P < 0.05) and reached a nadir on Day 14. There were decreases (P < 0.05) in seminiferous tubule diameter and testicular weight (relative to body weight) on Day 14. Testicular RNA was extracted, reverse-transcribed and cDNA used for PCR. Expression of genes Hspa1b (Hsp70) and Gpx1 had 7- and 10-fold increases (P < 0.001 for each) at 8 and 24 h, respectively, with Hspa1b remaining upregulated at 24 h, whereas StAR peaked at Day 14 (15-fold, P < 0.0001) and had returned to baseline on Day 21. Both Trp53 and Casp8 were upregulated (P < 0.05) on Day 14, whereas Bcl-2 was decreased (P < 0.05) on Days 7 and 14. In conclusion, acute mild heat stress severely reduced sperm quality and based on mRNA, there was upregulation of chaperone and antioxidant systems and Trp53-dependent intrinsic and extrinsic apoptotic pathways, with deleterious effects on sperm, spermatocytes and spermatids. These findings provided insights into the pathophysiology of heat stress and should contribute to development of evidence-based approaches to mitigate effects of testicular heating.

Short-term testicular warming under anesthesia causes similar increases in testicular blood flow in Bos taurus versus Bos indicus bulls, but no apparent hypoxia.

Bull testes must be 4-5 °C below body temperature, with testicular warming more likely to cause poor-quality sperm in Bos taurus (European/British) versus Bos indicus (Indian/zebu) bulls. Despite a long-standing dogma that testicular hyperthermia causes hypoxia, we reported that increasing testicular temperature in bulls and rams enhanced testicular blood flow and O2 delivery/uptake, without hypoxia. Our objective was to determine effects of short-term testicular hyperthermia on testicular blood flow, O2 delivery and uptake and evidence of testicular hypoxia in pubertal Angus (B. taurus) and Nelore (B. indicus) bulls (nine per breed) under isoflurane anesthesia. As testes were warmed from 34 to 40 °C, there were increases (P < 0.0001, but no breed effects) in testicular blood flow (mean ± SEM, 9.59 ± 0.10 vs 17.67 ± 0.29 mL/min/100 g, respectively), O2 delivery (1.79 ± 0.06 vs 3.44 ± 0.11 mL O2/min/100 g) and O2 consumption (0.69 ± 0.07 vs 1.25 ± 0.54 mL O2/min/100 g), but no indications of testicular hypoxia. Hypotheses that: 1) both breeds increase testicular blood flow in response to testicular warming; and 2) neither breed has testicular hypoxia, were supported; however, the hypothesis that the relative increase in blood flow is greater in Angus versus Nelore was not supported. Although these were short-term increases in testicular temperature in anesthetized bulls, results did not support the long-standing dogma that increased testicular temperature does not increase testicular blood flow and an ensuing hypoxia is responsible for decreases in motile, morphologically normal and fertile sperm.

Partial luteolysis during early diestrus in cattle downregulates VEGFA expression and reduces large luteal cell and corpus luteum sizes and plasma progesterone concentration.

Our objectives were to investigate potential changes in the size of steroidogenic large luteal cells (LLC) during partial luteolysis induced by a sub-dose of cloprostenol in early diestrus and to determine transcriptional variations in genes involved in corpus luteum (CL) functions. Cows were subjected to an Ovsynch protocol, with the time of the second GnRH treatment defined as Day 0 (D0). On D6, cows were randomly allocated into three treatments: Control (2 mL saline, im; n = 10), 2XPGF (two doses of 500 µg of cloprostenol, im, 2 h apart; n = 8) or 1/6PGF (single dose of 83.3 µg of cloprostenol, im; n = 10). Before treatments and every 8 h during the 48-h experimental period, blood samples were collected and CL volumes measured. Furthermore, two CL biopsies were obtained at 24 and 40 h post-treatment. The 1/6PGF treatment caused partial luteolysis, characterized by sudden decreases in plasma progesterone (P4) concentrations, luteal volume and LLC size, followed by increases (to pretreatment values) in P4 and luteal volume at 24 and 40 h post-treatment, respectively. However, at the end of the study, P4, luteal volume and LLC size were all significantly smaller than in Control cows. Temporally associated with these phenotypes, there was a lower mRNA abundance of VEGFA at 24 and 40 h, and ABCA1 at 24 h (P < 0.05). In conclusion, a sudden reduction in CL size during partial luteolysis induced by a sub-dose of PGF2α analog on day 6 of the estrous cycle was attributed to a reduction in LLC size, although these changes did not account for the entire phenomenon. In addition to its involvement in reducing CL size, decreased VEGFA mRNA abundance impaired CL development, resulting in a smaller luteal gland and lower plasma P4 concentrations compared to Control cows.

Testicular hyperthermia reduces testosterone concentrations and alters gene expression in testes of Nelore bulls.

Increased testicular temperature reduces sperm motility, morphology and fertility. Our objectives were to characterize effects of testicular hyperthermia (scrotal insulation) on acute testosterone concentrations and gene expression in Bos indicus testes. Nelore bulls (n = 20), ∼27 mo of age, 375 kg, scrotal circumference >31 cm, with ≥30% motile sperm, were allocated into four groups (n = 5/group): non-insulated (Control) and insulation removed after 12, 24, or 48 h. Immediately after insulation, intratesticular temperatures (needle thermocouples) were coolest in Control bulls and warmest in 48-h bulls (mean ± SEM, 35.28 ± 0.31 vs 38.62 ± 0.57 °C, P < 0.05). Bulls were castrated and testes recovered. Testicular testosterone concentrations were higher in Control versus 48-h bulls (3119 ± 973.3 and 295.5 ± 122.8 ng/g of tissue, respectively, P < 0.05). Total RNA was extracted, reverse transcribed and RT-qPCR done. For STAR, mRNA abundance decreased from Control to 48 h (1.14 + 0.32 vs 0.32 + 0.5, P < 0.05). For BCL2, expression decreased from Control to 24 h (1.00 + 0.07 vs 0.70 + 0.12, P < 0.05), but then rebounded. In addition, GPX1 had a 70% increase (P < 0.05) at 48 h, whereas HSP70 had a 34-fold increase (P < 0.05) at 12 h and 2- and 14-fold increases (P < 0.05) at 24 and 48 h, respectively. HSF1, BAX, P53 and CASP 8 remained unchanged. Downregulation of STAR, critical in androgen production, was consistent with reduced testosterone concentrations, whereas increased GPX1 enhanced testicular antioxidative capability. Huge increases in HSP70 conferred protection again apoptosis and cell destruction, whereas reduced BCL2 promoted apoptosis. These findings provided novel insights into acute tissue responses (testosterone and gene activity) to testicular hyperthermia in B. indicus bulls.

Elevated testicular temperature modulates expression patterns of sperm proteins in Holstein bulls.

The objective of this study was to determine the effects of elevated testicular temperature on the expression patterns of sperm proteins in bulls. Ejaculates were collected from sexually mature Holstein bulls (n = 6) twice weekly for 10 weeks. Testicular temperature was elevated in all bulls by scrotal insulation for 72 consecutive hours during Week 2. Triton X-100 extracts of sperm proteins were prepared and subjected to SDS-PAGE. Sperm proteins in the 110 kDa range decreased from pre-thermal insult to Day 28 (start of thermal insult = Day 0), concurrent with decreases in sperm concentration, motility, and morphology, and subsequently increased, approaching pre-thermal insult values by Day 40. Based on mass spectrometry, this band was comprised of angiotensin converting enzyme (ACE), Hexokinase-1, and the alpha-4 subunit of Na(+)/K(+)ATPase. Changes in the expression patterns of these proteins were confirmed by immunoblotting, including the use of a custom antibody against the alpha-4 subunit of Na(+)/K(+)ATPase (testis-specific isoform). Furthermore, a 25 kDa sperm protein (identified as tissue inhibitor of metalloproteinase-2; TIMP-2), had a low expression level in pre-thermal insult samples, increased to Day 28 of post-thermal insult, and subsequently decreased to the pre-thermal insult level by the end of the experimental period. In conclusion, we identified proteins that may serve as molecular markers of impaired sperm function due to elevated testicular temperature, with important implications for fertility predictions. To our knowledge, this is the first report of the identification of a testis-specific isoform of Na(+)/K(+)ATPase in bovine sperm.