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.

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).

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.

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.

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.

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.

Injectable or transdermal flunixin meglumine improves pregnancy rates in embryo transfer recipient beef cows without altering returns to estrus.

OBJECTIVES: were to determine effects of 1) injectable or transdermal flunixin meglumine (FM) at embryo transfer (ET) compared to an untreated control group on pregnancy per ET (P/ET; ∼35 d after ET); 2) embryo and recipient factors on P/ET; 3) FM on hormone concentrations; and 4) FM on returns to estrus. Angus-cross beef cows (n = 1145) at five locations were scored for body condition (BCS; 1-9) and temperament (0 or 1) and given Select-Synch + CIDR. Recipient cows with a corpus luteum (CL) ≥1.5 cm received a frozen-thawed embryo 7 d after estrus and were concurrently given 1.1 mg/kg injectable FM im (INJFM; n = 384), 3.3 mg/kg transdermal FM pour on (TDFM; n = 388), or nothing (CON group; n = 373). Blood samples were collected at ET and 7 d later (60 cows). Accounting for temperament (P < 0.05), ET difficulty score (1-3, easy to difficult; P < 0.01), treatment by temperament (P < 0.001) and treatment by embryo quality (P < 0.05), FM treatments affected P/ET (P < 0.05). The P/ET for cows given INJFM [62.8% (241/384)] or TDFM [58.7% (228/388)] were not different (P = 0.26), but they were greater (P = 0.01 and P = 0.04, respectively) than P/ET for controls [51.2% (191/373)]. The P/ET was greater for calm versus excitable cows, 60.2 (463/769) and 52.4% (197/376), respectively (P < 0.01) and was lower for difficulty score 3 [49.2% (156/317)] compared to score 1 [62.7% (254/405; P < 0.001) or score 2 [59.1% (250/423; P < 0.01)]. There was no effect (P > 0.1) of cow age, BCS, or stage of embryo development on P/ET. Pregnancy rates for embryo quality grade 1 (excellent/good) and grade 2 (fair) were 60.4% (314/520) and 55.4% (346/625), respectively (P > 0.05). Percentages of non-pregnant recipient cows in estrus from Days 18-26 did not differ among treatment groups (P > 0.1). Control cows had lower progesterone concentrations and greater substance-P, PGFM and 8-isoprostane PGF2α concentrations at 7 d after ET compared to FM-treated cows (P < 0.05). In conclusion, injectable or transdermal FM improved pregnancy rates in ET recipients, without affecting nonpregnant cows return to estrus.

Hyperthermia is more important than hypoxia as a cause of disrupted spermatogenesis and abnormal sperm.

We tested the hypothesis that hypoxia replicates effects of hyperthermia on reducing number and quality of sperm produced, whereas hyperoxia mitigates effects of hyperthermia. Forty-eight CD-1 mice (∼50 d old), inspired air with 13, 21, or 95% O2 and were exposed to ambient temperatures of 20 or 36 °C (3 × 2 factorial, six groups) twice for 12 h (separated by 12 h at 20 °C and 21% O2), with euthanasia 14 or 20 d after first exposure. Combined for both post-exposure intervals, there were primarily main effects of temperature; mice exposed to 20 vs 36 °C had differences in testis weight (110.2 vs 96.9 mg, respectively; P < 0.0001), daily sperm production (24.7 vs 21.1 × 106 sperm/g testes, P < 0.03), motile sperm (54.5 vs 41.5%, P < 0.002), morphologically normal sperm (59.9 vs 45.4%, P < 0.002), morphologically abnormal heads (7.3 vs 22.0%, P < 0.0001), seminiferous tubule diameter (183.4 vs 176.3 µm, P < 0.004) and altered elongated spermatids (2.2 vs 15.9, P < 0.001). Increasing O2 (from 13 to 95%) affected morphologically abnormal heads (15.4, 10.8 and 17.6%, respectively; P < 0.03), seminiferous tubule diameter (175.7, 185.6 and 178.4 µm, P < 0.003) and total altered spermatids (8.3, 3.3 and 15.2, P < 0.05). Our hypothesis was not supported; hypoxia did not replicate effects of hyperthermia with regards to reducing number and quality of sperm produced and hyperoxia did not mitigate effects of hyperthermia. We concluded that hyperthermia per se and not secondary hypoxia was the fundamental cause of heat-induced effects on spermatogenesis and sperm. These findings are of interest to develop evidence-based efforts to mitigate effects of testicular hyperthermia, as efforts should be focused on hyperthermia per se and not on hyperthermia-induced hypoxia.

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.

Sperm and seminal plasma proteomics of high- versus low-fertility Holstein bulls.

Sperm are highly specialized compartmentalized cells, with unique compositional, morphological and functional properties, including a plasma membrane that undergoes dynamic protein remodeling and surface modifications. Seminal plasma is a highly complex biological fluid containing proteins, amino acids, enzymes, fructose and other carbohydrates, lipids, major minerals and trace elements. Seminal plasma proteins are involved in regulation of osmotic pressure and pH of seminal plasma, transport of ions, lipid and hormones. The objective was to compare sperm and seminal plasma proteomes of bulls with differing fertility and to relate differences to biological processes. Semen was collected from bulls with high or low fertility (4 bulls in each category). Sperm and seminal plasma proteins were isolated, purified, subjected to 2-D gel electrophoresis, protein identification and ontology. In sperm and seminal plasma, binder of sperm proteins (BSP)-1, -3 and -5, and spermadhesin-1, ALB, TIMP, AKI and PEBP1 were higher for high-versus low-fertility bulls (P < 0.05), whereas proteins CLU, CCT5 and 8, ELSPbP1, and PSMA6 were more abundant in sperm and seminal plasma of low- versus high-fertility bulls (P < 0.05). Further, HSP90, ZFP34, IFNRF4, BCL62, NADHD, TUBB3 and Histone H1 were in greater abundance in sperm of high- compared with low-fertility bulls. The two key biological processes of proteins differentially expressed in high- and low-fertility bulls were metabolic processes and biological regulation. The most prominent molecular functions for proteins that differed are binding, catalytic and receptor activities. The main cellular components for proteins that differed are cellular, extracellular, and plasma membrane. Since protein content differed in high- versus low-fertility bulls, we inferred that the efficiency of associated sperm functions that are necessary for fertility may also differ between high- and low-fertility semen. In conclusion, differences between high- and low-fertility bulls regarding abundance of sperm and seminal plasma proteins likely contributed to differences in fertility.

Increased testicular blood flow maintains oxygen delivery and avoids testicular hypoxia in response to reduced oxygen content in inspired air.

Despite a long-standing assertion that mammalian testes operate near hypoxia and increased testicular temperature causes frank hypoxia, we have preliminary evidence that changes are due to hyperthermia per se. The objective was to determine how variations in inspired oxygen concentration affected testicular blood flow, oxygen delivery and extraction, testicular temperature and lactate production. Eight rams were maintained under general anesthesia, with successive decreases in oxygen concentration in inspired air (100, 21 and 13%, respectively). As oxygen concentration decreased from 100 to 13%, there were increases in testicular blood flow (9.6 ± 1.7 vs 12.9 ± 1.9 ml/min/100 g of testis, P < 0.05; mean ± SEM) and conductance (normalized flow; 0.46 ± 0.07 to 1.28 ± 0.19 ml/min/mm Hg/100 g testis (P < 0.05). Increased testicular blood flow maintained oxygen delivery and increased testicular temperature by ~1 °C; this increase was correlated to increased testicular blood flow (r = 0.35, P < 0.0001). Furthermore, oxygen utilization increased concomitantly and there were no significant differences among oxygen concentrations in blood pH, HCO3- or base excess, and no effects of venous-arterial differences in lactate production. In conclusion, under acute hypoxic conditions, testes maintained oxygen delivery and uptake by increasing blood flow and oxygen extraction, with no evidence of anaerobic metabolism. However, additional studies are needed to determine longer-term responses and potential evidence of anaerobic metabolism at the molecular level.

Review: Testicular vascular cone development and its association with scrotal thermoregulation, semen quality and sperm production in bulls.

Several structural and functional features keep bull testes 2°C to 6°C below body temperature, essential for the production of morphologically normal, motile and fertile sperm. The testicular vascular cone (TVC), located above the testis, consists of a highly coiled testicular artery surrounded by a complex network of small veins (pampiniform plexus). The TVC functions as a counter-current heat exchanger to transfer heat from the testicular artery to the testicular vein, cooling blood before it enters the testis. Bulls with increased TVC diameter or decreased distance between arterial and venous blood, have a greater percentage of morphologically normal sperm. Both the scrotum and testes are warmest at the origin of their blood supply (top of scrotum and bottom of testis), but they are cooler distal to that point. In situ, these opposing temperature gradients result in a nearly uniform testicular temperature (top to bottom), cooler than body temperature. The major source of testicular heat is blood flow, not testicular metabolism. High ambient temperatures have less deleterious effects on spermatogenesis in Bos indicus v. Bos taurus bulls; differences in TVC morphology in B. indicus bulls confer a better testicular blood supply and promote heat transfer. There is a long-standing paradigm that testes operate on the brink of hypoxia, increased testicular temperature does not increase blood flow, and the resulting hypoxia reduces morphologically normal and motile sperm following testicular hyperthermia. However, in recent studies in rams, either systemic hypoxia or increased testicular temperature increased testicular blood flow and there were sufficient increases in oxygen uptake to prevent tissue hypoxia. Therefore, effects of increased testicular temperature were attributed to testicular temperature per se and not to secondary hypoxia. There are many causes of increased testicular temperature, including high ambient temperatures, fever, increased recumbency, high-energy diets, or experimental insulation of the scrotum or the scrotal neck. It is well known that increased testicular temperatures have adverse effects on spermatogenesis. Heat affects all germ cells and all stages of spermatogenesis, with substantial increases in temperature and/or extended intervals of increased testicular temperature having the most profound effects. Increased testicular temperature has adverse effects on percentages of motile, live and morphologically normal sperm. In particular, increased testicular temperature increases the percentage of sperm with abnormal morphology, particularly head defects. Despite differences among bulls in the kind and percentage of abnormal sperm, the interval from increased testicular temperature to the emergence of specific sperm defects is consistent and predictable. Scrotal surface temperatures and structural characteristics of the testis and TVC can be assessed with IR thermography and ultrasonography, respectively.

Arterial blood flow is the main source of testicular heat in bulls and higher ambient temperatures significantly increase testicular blood flow.

Two experiments were done in bulls to determine: total testicular blood flow, testis oxygenation and heat, and effects of ambient temperature on testicular temperatures and blood flow. In Experiment 1, arterial blood flow to testes and testicular oxygenation and heat were determined in Angus bulls (n = 8). Blood temperature and hemoglobin O2 saturation were both greater (P < 0.0001) in the testicular artery than in the testicular vein (39.2 ±â€¯0.2 vs 36.9 ±â€¯0.4 °C and 95.3 ±â€¯0.7 vs 42.0 ±â€¯5.8%, respectively; mean ±â€¯SEM). Based on testicular blood flow of 12.4 ±â€¯1.1 mL/min and an arterial-venous temperature differential of 2.3 °C, blood contributed 28.3 ±â€¯5.1 cal/min of heat to the testis, whereas heat produced by testicular metabolism was estimated at 5.8 ±â€¯0.8 cal/min (based on O2 consumption of 1.2 ±â€¯0.2 mL/min). In Experiment 2, effects of three ambient temperatures (5, 15 and 35 °C) on testicular blood flow and temperatures were determined in 20 Angus bulls. At 35 versus 5 °C, there was greater testicular blood flow (8.2 ±â€¯0.9 versus 4.9 ±â€¯0.7 mL/min/100 g of testicular tissue, P < 0.05), and higher scrotal subcutaneous and intratesticular temperatures (P < 0.01). In conclusion, arterial blood flow was the main source of testicular heat, testes were close to hypoxia, and increased ambient temperature significantly increased scrotal subcutaneous and intratesticular temperatures, as well as testicular blood flow. These studies gave new insights into scrotal/testicular thermoregulation in bulls; they confirmed that testes are nearly hypoxic, but challenged the long-standing paradigm that testicular blood flow does not increase when testes become warmer.

Knowledge gaps that hamper prevention and control of Mycobacterium avium subspecies paratuberculosis infection.

In the last decades, many regional and country-wide control programmes for Johne's disease (JD) were developed due to associated economic losses, or because of a possible association with Crohn's disease. These control programmes were often not successful, partly because management protocols were not followed, including the introduction of infected replacement cattle, because tests to identify infected animals were unreliable, and uptake by farmers was not high enough because of a perceived low return on investment. In the absence of a cure or effective commercial vaccines, control of JD is currently primarily based on herd management strategies to avoid infection of cattle and restrict within-farm and farm-to-farm transmission. Although JD control programmes have been implemented in most developed countries, lessons learned from JD prevention and control programmes are underreported. Also, JD control programmes are typically evaluated in a limited number of herds and the duration of the study is less than 5 year, making it difficult to adequately assess the efficacy of control programmes. In this manuscript, we identify the most important gaps in knowledge hampering JD prevention and control programmes, including vaccination and diagnostics. Secondly, we discuss directions that research should take to address those knowledge gaps.

Flunixin meglumine improves pregnancy rate in embryo recipient beef cows with an excitable temperament.

Objectives were to determine effects of: 1) handling temperament and administration of flunixin meglumine, an inhibitor of prostaglandin F2a (PGF2a) synthesis, given at the time of embryo transfer, on pregnancy rates in beef cattle embryo transfer recipients; 2) handling temperament and flunixin meglumine on peripheral concentrations of progesterone, cortisol, substance-P, prostaglandin F metabolites (PGFM, (13,14-dihydro-15-keto-PGF2a) and isoprostane 8-epi PGF2a; and 3) flunixin meglumine treatment on proportion of non-pregnant recipient cows returning to estrus within an expected interval. Angus cross beef cows (n = 710) at 7 locations were assigned a body condition score (BCS: 1, emaciated; 9, obese) and a temperament score [0, calm, slow chute exit; walk (n = 352), 1, excited, fast chute exit; jump, trot or run (n = 358)] and were synchronized with Select-Synch with a controlled internal drug release (CIDR) protocol. Estrus detection aids were applied at CIDR removal and cows were observed thrice daily for estrus until 72 h. Recipient cows that expressed estrus and had a corpus luteum received a frozen-thawed embryo on Day 7 after estrus. At the time of transfer, recipient cows were randomly allocated to receive 10 mL of flunixin meglumine im, immediately after transfer (n = 365) or served as an untreated control (n = 345). In a subset of cows (n = 80), ovarian ultrasonography was performed on the day of embryo transfer to determine corpus luteum volume and blood samples were collected twice, at the time of embryo transfer and 7 d later. All cows received estrus detection aids again on Day 14 (7 d after embryo transfer) and were observed for estrus twice daily until Day 24. Accounting for treatment (P > 0.1), embryo transfer difficulty score (P < 0.1), temperament by treatment interaction (P < 0.05), recipient cows with calm temperament had a higher pregnancy rate compared to those with an excited temperament [59.4 (209/352) vs 51.7% (185/358)]. The pregnancy rate for excitable cows without flunixin meglumine was lower (46.3% 81/175) compared to excitable cows that did received flunixin meglumine [56.8% (104/183)], and calm cows that did [59.3% (108/182)] or did not [59.4% (104/170)] receive flunixin meglumine. Proportions of non-pregnant recipient cows returning to estrus on Days 18-24 were not different between flunixin meglumine and control groups, 87.6% (134/153) and 84.0% (137/163), respectively (P > 0.1). At the time of embryo transfer and 7 d later, there were moderate to strong correlations among circulating concentrations of progesterone, cortisol, substance-P, PGFM and isoprostane 8-epi PGF2a. Among excitable cows, progesterone concentrations were lower and cortisol, substance-P, PGFM and isoprostane 8-epi PGF2a concentrations were greater for cows in the control group compared to cows that received flunixin meglumine. In conclusion, administration of flunixin meglumine improved pregnancy rates in excitable recipient cows following embryo transfer without affecting the proportion of non-pregnant cows returning to estrus.

Predictors and impacts of colostrum consumption by 4h after birth in newborn beef calves.

Newborn beef calf vigor is a vital survival characteristic that promotes timely colostrum consumption. Modified APGAR scores intended to identify compromised calves are not widely adopted due to impracticality, inconsistent associations with blood gas disturbances, and unclear recommendations for intervention. The objectives were to: (1) determine differences in at-birth blood parameters between calves that were successful in colostrum consumption by 4h (CC4) compared to those that failed; (2) develop a Beef Calf Vigor Assessment by determining calving characteristics and clinical examination parameters associated with CC4; and (3) evaluate impacts of failed CC4 on transfer of passive immunity and treatment risk. As a predictor for CC4, suckle reflex had the highest specificity (98%), whereas calving ease had the highest sensitivity (89%). Calves with a weak suckle reflex had 41.6 (95% CI: 7.4-787.5) times greater odds of failed CC4 compared to calves with a strong suckle reflex (P<0.0001). Calves with failed CC4 had lower serum IgG (P=0.01), had lower odds of acquiring optimal passive immunity (>24g/L serum IgG; OR=6.4, 95% CI: 1.2-34.4; P=0.02), and higher odds of being treated (OR=2.8, 95% CI: 1.1-7.4; P=0.03) than those that succeeded. Measuring suckle reflex in combination with calving ease was a quick and easy method to assess newborn beef calf vigor. The value of this Beef Calf Vigor Assessment was further emphasized by the negative impacts of failed CC4 on transfer of passive immunity and pre-weaning health.

Testis-specific isoform of Na/K-ATPase (ATP1A4) regulates sperm function and fertility in dairy bulls through potential mechanisms involving reactive oxygen species, calcium and actin polymerization.

Traditional bull breeding soundness evaluation (BBSE) eliminates bulls that are grossly abnormal; however, bulls classified as satisfactory potential breeders still vary in field fertility, implying submicroscopic differences in sperm characteristics. The testis-specific isoform of Na/K-ATPase (ATP1A4) is involved in regulation of sperm motility and capacitation in bulls through well-established enzyme activity and signaling functions. The objective was to determine ATP1A4 content, activity and their relationship to post-thaw sperm function and field fertility, using semen samples from low-fertility (LF) and high-fertility (HF) Holstein bulls (n = 20 each) with known FERTSOL rates (measure of field fertility, based on non-return rate). Frozen-thawed sperm from HF bulls had increased ATP1A4 content and activity compared to LF bulls. Furthermore, post-thaw sperm from HF bulls had increased tyrosine phosphorylation, ROS, F-actin content, and low intracellular calcium compared to LF bulls. Subsequent incubation of HF bull sperm with ouabain (a specific ligand of Na/K-ATPase) further augmented the post-thaw increase in tyrosine phosphorylation, ROS production, and F-actin content, whereas the increase in intracellular calcium was still low compared to LF bull sperm. ATP1A4 content and activity, ROS, F-actin and calcium were significantly correlated with fertility. In conclusion, we inferred that ATP1A4 content and activity differed among dairy bulls with satisfactory semen characteristics and that ATP1A4 may regulate sperm function through mechanisms involving ROS, F-actin and calcium in frozen-thawed sperm of HF and LF dairy bulls.

Clinical indicators of blood gas disturbances, elevated L-lactate concentration and other abnormal blood parameters in newborn beef calves.

Blood gas disturbances, commonly resulting from dystocia, are associated with failed transfer of passive immunity, morbidity and mortality in newborn calves. Modified APGAR scores intended to identify compromised calves are not widely adopted due to lack of practicality and inconsistent associations with blood parameters. The objective of this study was to determine clinical indicators of acidemia in newborn beef calves. Blood parameters at 10 min and 24 h after birth were compared to at-birth clinical examination parameters and calving characteristics in 77 commercial beef calves. There were no associations between heart rate or respiratory rate and blood pH or blood L-lactate concentration (LAC; r < 0.25); however, LAC was highly correlated with blood pH (r, -0.86). Abnormal mucous membrane color (red, white or blue) was associated with increased LAC (P = 0.002) but not decreased blood pH (P = 0.07). Abnormal results for tests of muscle tonicity and reflexes, namely inability to completely withdraw the tongue when pinched and a weak suckle reflex, were associated with decreased blood pH and increased LAC (P <0.05). Calves born to primiparous dams or from an assisted calving also had decreased blood pH and increased LAC (P <0.05). Differences in blood pH between at-birth categories resolved for all parameters by 24 h after birth, except for tongue withdrawal (P= 0.04). In conclusion, traditional APGAR parameters, heart rate, respiratory rate and mucous membrane color were not useful for the identification of acidemic calves; however, tongue withdrawal, calving ease, and parity should be included in such an assessment. Utilizing hand-held LAC meters may be a practical method to quickly identify compromised calves with acidemia in a field setting.