T. J. Safranski

Differential expression of porcine sperm microRNAs and their association with sperm morphology and motility.

MicroRNAs (miRNAs) are involved in nearly every biological process examined to date, but little is known of the identity or function of miRNA in sperm cells or their potential involvement in spermatogenesis. The objective was to identify differences in miRNA expression between normal porcine sperm samples and those exhibiting high percentages of morphological abnormalities or low motility. Quantitative RT-PCR was performed on sperm RNA to compare expression levels of 10 specific miRNAs that are predicted to target genes that code for proteins involved in spermatogenesis, sperm structure, motility, or metabolism. There were increases in the expression of four miRNAs, let-7a, -7d, -7e, and miR-22, in the abnormal group (P < 0.05), whereas miR-15b was decreased compared to controls (P < 0.05). Two miRNAs, let-7d and let-7e, were increased in the low motility group when compared to controls (P < 0.05). Bioinformatic analyses revealed that messenger RNA targets of the differentially expressed miRNAs encode proteins previously described to play roles in sperm function. Although the precise role of miRNA in sperm remains to be determined, their changes as associated with morphology and motility signify a critical biological function. Perhaps they are remnants of spermatogenesis, stored for a later role in fertilization, or are delivered to the oocyte to influence early embryonic development. Although there is no single cause of male infertility, the identification of miRNAs associated with sperm motility, structural integrity, or metabolism could lead to the development of a microarray or real time-based diagnostic assay to provide an assessment of male fertility status.

An update on North American boar stud practices.

This survey included 44 boar studs from Canada and the USA with a total of approximately 10,000 boars. Studs with 51-500 boars accounted for 84% of respondents. More than 90% of boars were housed in stalls. Evaporative and mechanical cooling systems predominated and boars were typically fed based on body condition. The predominant age of boars was 1-2 years with annual culling rates between 20 and 70%. The primary reasons for culling included genetic improvement, semen quality and feet and leg issues. Collection occurred commonly on Mondays and Thursdays and boars were rested 3-7 days between collections. The average sperm produced per boar per week was 51-150 billions and resulted in 21-40 doses per boar per week. Most studs collected boars using double gloves and disposable cups or liners and used pre-warmed containers. Ejaculate pooling was practiced by >60% of studs. Evaluation of semen for motility was performed with 0-5min of warming in extender with viewing at 100-400x magnification. Concentration estimation occurred by photometer and CASA for 88% of studs. Ejaculate discard occurred for reasons of poor motility, abnormal sperm and bacteria. Most studs retained extended samples for 3-7 days for quality control. Discard rates were most common between 1 and 10% and were related to individual boar and season. Doses of semen contained 2-4 billion sperms, with final sperm numbers adjusted for fertile sperm and packaged as doses in tubes and bags with 60-100mL.

Effects of in utero heat stress on postnatal body composition in pigs: I. Growing phase.

Environmentally induced heat stress (HS) negatively influences production variables in agriculturally important species. However, the extent to which HS experienced in utero affects nutrient partitioning during the rapid lean tissue accretion phase of postnatal growth is unknown. Study objectives were to compare future whole-body tissue accretion rates in pigs exposed to differing in utero and postnatal thermal environments when lean tissue deposition is likely maximized. Pregnant sows were exposed to thermoneutral (TN; cyclical 15°C nighttime and 22°C daytime; n = 9) or HS (cyclical 27°C nighttime and 37°C daytime; n = 12) conditions during their entire gestation. Twenty-four offspring from in utero TN (IUTN; n = 6 gilts and 6 barrows; 30.8 ± 0.2 kg BW) and in utero HS (IUHS; n = 6 gilts and 6 barrows; 30.3 ± 0.2 kg BW) were euthanized as an initial slaughter group (ISG). Following the ISG, 48 pigs from IUTN (n = 12 gilts and 12 barrows; 34.1 ± 0.5 kg BW) and IUHS (n = 12 gilts and 12 barrows; 33.3 ± 0.3 kg BW) were exposed to constant HS (34.1 ± 2.4°C) or TN (21.5 ± 2.0°C) conditions until they reached 61.5 ± 0.8 kg BW, at which point they were sacrificed and their whole-body composition was determined. Homogenized carcasses were analyzed for N, crude fat, ash, water, and GE content. Data were analyzed using PROC MIXED in SAS 9.3. Rectal temperature and respiration rate increased (P < 0.01) during postnatal HS compared to TN (39.4 vs. 39.0°C and 94 vs. 49 breaths per minute, respectively). Regardless of in utero environment, postnatal HS reduced (P < 0.01) feed intake (2.06 vs. 2.37 kg/d) and ADG (0.86 vs. 0.98 kg/d) compared to TN conditions. Postnatal HS did not alter water, protein, and ash accretion rates but reduced lipid accretion rates (198 vs. 232 g/d; P < 0.04) compared to TN-reared pigs. In utero environment had no effect on future tissue deposition rates; however, IUHS pigs from the ISG had reduced liver weight (P < 0.04; 17.9%) compared to IUTN controls. In summary, postnatal HS reduced adipose tissue accretion rates, but IUHS did not appear to impact either lean or adipose tissue accretion during this specific growth phase.

Gestational heat stress alters postnatal offspring body composition indices and metabolic parameters in pigs.

The study objectives were to test the hypothesis that heat stress (HS) during gestational development alters postnatal growth, body composition, and biological response to HS conditions in pigs. To investigate this, 14 first parity crossbred gilts were exposed to one of four environmental treatments (TNTN, TNHS, HSTN, or HSHS) during gestation. TNTN and HSHS dams were exposed to thermal neutral (TN, cyclical 18–22°C) or HS conditions (cyclical 28–34°C) during the entire gestation, respectively. Dams assigned to HSTN and TNHS treatments were heat-stressed for the first or second half of gestation, respectively. Postnatal offspring were exposed to one of two thermal environments for an acute (24 h) or chronic (five weeks) duration in either constant TN (21°C) or HS (35°C) environment. Exposure to chronic HS during their growth phase resulted in decreased longissimus dorsi cross-sectional area (LDA) in offspring from HSHS and HSTN treated dams whereas LDA was larger in offspring from dams in TNTN and TNHS conditions. Irrespective of HS during prepubertal postnatal growth, pigs from dams that experienced HS during the first half of gestation (HSHS and HSTN) had increased (13.9%) subcutaneous fat thickness compared to pigs from dams exposed to TN conditions during the first half of gestation. This metabolic repartitioning towards increased fat deposition in pigs from dams heat-stressed during the first half of gestation was accompanied by elevated blood insulin concentrations (33%; P = 0.01). Together, these results demonstrate HS during the first half of gestation altered metabolic and body composition parameters during future development and in biological responses to a subsequent HS challenge.

Effects of a controlled heat stress during late gestation, lactation, and after weaning on thermoregulation, metabolism, and reproduction of primiparous sows

Heat stress (HS) causes seasonal infertility in sows and decreases reproductive efficiency. The objective was to examine thermoregulation, metabolic responses, and reproduction in sows exposed to HS or thermoneutral (TN) conditions during different phases of a production cycle (gestation, lactation, and breeding). Fifty-eight first-parity Landrace (n = 26) or Landrace × Large White F1 (n = 32) sows were rotated through environmental chambers for 57 d beginning in late gestation. The ambient temperature sequences included either TN (18°C to 20°C) or HS (24°C to 30°C) for each production phase with the following treatment groups: TN-TN-TN (n = 15), TN-HS-TN (n = 14), HS-TN-HS (n = 14), and HS-HS-HS (n = 15) for gestation-farrowing-breeding (20, 24, and 13 d, respectively). Regardless of the temperature treatment, rectal temperatures were greater (P < 0.001) during lactation (39.36°C ± 0.01°C) than during the gestation (38.27°C ± 0.01°C) or the breeding period (38.77°C ± 0.01°C). The increase in rectal temperature (P < 0.001) and respiration rate (P < 0.001) in response to the HS was greatest during lactation. There was an effect of day (P < 0.001) on serum IGF-1 and insulin concentrations because both insulin and IGF-1 increased after farrowing. Compared with HS sows, the TN sows had greater feed intake (P < 0.001) and greater serum concentrations of insulin (early lactation; P < 0.05) and IGF-1 (late lactation; P < 0.05) when they were lactating. The effects of HS on sow BW, back fat, and loin eye area were generally not significant. Average BW of individual piglets at weaning was approximately 0.5 kg lighter for the sows in the HS farrowing room (P < 0.05). Weaning-to-estrus interval, percentage sows inseminated after weaning, subsequent farrowing rate, and subsequent total born were not affected by treatment. In summary, regardless of ambient temperature, sows undergo pronounced and sustained changes in rectal temperature when they transition through gestation, lactation, weaning, and rebreeding. The effects of HS on rectal temperature, respiration rate, feed intake, and metabolic hormones were greatest during lactation. The controlled HS that we imposed affected piglet weaning weight, but rebreeding and subsequent farrowing performance were not affected.

Factors affecting follicular populations on Day 3 postweaning and interval to ovulation in a commercial sow herd.

Sows (n=146) in a commercial herd were studied to determine factors affecting follicular populations and interval to ovulation after weaning. Ovaries were examined daily by ultrasonography beginning on Day 3 postweaning and twice daily from Day 4.5 until ovulation. Ovarian images were recorded on videotape on Day 3 postweaning and follicles were counted. Subsequent ultrasounds were used to determine time of ovulation. Sows with short weaning to ovulation intervals (<or=6.5 days) had follicular populations on Day 3 postweaning that were more advanced (comprised of follicles with greater diameter) when compared to sows with long (>or=9 days) weaning to ovulation intervals (P<0.001). Follicular populations in sows with intermediate (7-8.5 days) intervals to ovulation were intermediate in diameter when compared to sows with short or long intervals to ovulation. Parity and body condition score (BCS) affected interval to ovulation; first parity and low body condition sows had longer intervals to ovulation (P<0.001 and 0.05, respectively). The longer intervals to ovulation in first parity and low body condition sows were associated with lesser follicular diameters on Day 3 after weaning. We conclude that follicular populations measured by ultrasonography on Day 3 after weaning were different for sows with different intervals to ovulation. Furthermore, production factors (i.e. parity and BCS) known to influence interval to ovulation were associated with differences in follicular growth within the first 3 days after weaning in sows.

An analysis of survey data by size of the breeding herd for the reproductive management practices of North American sow farms

A survey was performed to assess whether reproductive management differed among small-sized (Sm, <500 sows), medium-sized (M, 501 to 2,000 sows), and large-sized (Lg, 2,001 to 8,000 sows) farms (n=113). Farms with 501 to 4000 sows/barn were most frequent with sows kept in stalls on 90% of farms. More Lg farms (P<0.05) functioned as breed to wean and more Sm and M as farrow to finish. More Sm and Lg farms weaned at >21 d, whereas M farms were more likely to wean at 18 to 21 d (P<0.05). More Lg farms had farrowing rates above 89% than Sm and M farms (P<0.05), and culling rates above 40% were more frequent on M and Lg farms than on S. On M and Lg farms, sows were bred in larger batches, using lower person to sow ratios, and with more people required than on Sm farms (P<0.05). More (P<0.05) M and Lg farms spent time moving sows and on records, but hours devoted to estrous detection, breeding, and other tasks did not differ among farms (P>0.10). More M and Lg farms used more boars for estrus detection, rotated boars, and controlled boar movement than Sm farms (P<0.05). Farm size also influenced semen sourcing, number of doses received, and frequency of semen delivery (P<0.05). More M and Lg farms performed AI in the presence of a boar, left the AI rod in after AI, checked for returns, and diagnosed pregnancy than Sm farms (P<0.05). Start of boar exposure after weaning began on 69% of farms within 2 d, occurring most often in the AM, but with exposure times varying from 1 to 5 min/sow. Semen was thermally protected for 50% of farms receiving shipments, and semen storage was consistent among farms. For AI, service occurred within minutes to hours after detection of estrus on 61% of farms. During AI, procedures such as back-pressure were required, whereas techniques such as hands-free AI were prohibited on most farms. Sow movement was allowed only once at 4 wk after breeding on 50% of farms, and pregnancy diagnosis occurred at 3 to 5 wk on 78% of farms. Most sows were allowed ≥1 chance for breeding after conception failure before culling. Incidence of fail to farrow was <5% and litter size was 10 to 13 pigs on >82% of farms. Summer infertility was observed on 69% of farms with estrus and pregnancy failures the leading causes. Over 70% of farms reported a technician effect on fertility. These results suggest that reproductive management of farms in key areas related to weaning, breeding, gestation, and labor use could be a source of variation in reproductive performance.

Carcass composition of market weight pigs subjected to heat stress in utero and during finishing.

Objectives were to investigate the effects of prolonged gestational and/or postnatal heat stress on performance and carcass composition of market weight pigs. Pregnant gilts were exposed to gestational heat stress (GHS, 28°C to 34°C, diurnal) or thermal neutral (18°C to 22°C, diurnal) conditions during the entire gestation or during the first or second half of gestation. At 14 wk of age (58 ± 5 kg), barrows were housed in heat stress (32°C, HS) or thermal neutral (21°C, TN) conditions. Feed intake and BW were recorded weekly, and body temperature parameters were monitored twice weekly until slaughter (109 ± 5 kg). Organs were removed and weighed, and loin eye area (LEA) and back fat thickness (BF) were measured after carcass chilling. Carcass sides were separated into lean, separable fat, bone, and skin components and were weighed. Moisture, lipid, and protein content were determined in the LM at the 10th rib. Data were analyzed using a split plot with random effect of dam nested within gestational treatment. Carcass measurements included HCW as a covariate to control for weight. Planned orthogonal contrast statements were used to evaluate the overall effect of GHS in the first half, second half, or any part of gestation. Gestational heat stress did not alter postnatal performance or most body temperature parameters (P > 0.10). However, ADFI in the finishing period was increased (P < 0.05) in response to GHS, particularly in pigs receiving GHS in the first half of gestation. Gestational heat stress during the first half of gestation decreased head weight as a percent of BW (P = 0.02), whereas GHS in the second half of gestation decreased bone weight as a percent of BW (P = 0.02). Heat stress reduced ADG, BW, and HCW (P < 0.0001). Lean tissue was increased in HS pigs on both a weight and percentage basis (P < 0.0001), but LEA was similar to TN carcasses (P = 0.38). Carcasses from HS barrows also had less carcass separable fat (P < 0.01) and tended to have less BF (P = 0.06) compared with those from TN barrows, even after controlling for HCW. However, percent intramuscular fat did not differ between treatments (P = 0.48). The LM from HS carcasses had a greater moisture to protein ratio (P = 0.04). HS barrows also had decreased heart (P < 0.001) and kidney (P < 0.0001) as a percent of BW compared with TN pigs. In summary, GHS may affect head and bone development, subsequently affecting carcass composition. Chronic HS during finishing results in longer times to reach market weight and a leaner carcass once market weight is achieved.

Altered epididymal sperm maturation and cytoplasmic droplet migration in subfertile male Alox15 mice.

Mammalian spermatozoa complete their morphogenesis and acquire their fertilizing potential in the epididymis. Prominent among the hallmarks of epididymal sperm maturation is the proximal-distal migration of the cytoplasmic droplet (CD), the last remnant of the spermatogenic cell cytoplasm, down the sperm flagellum. Failure to shed the CD has been associated with male infertility. Because of the presence of the organelle degradation enzyme 15-lipoxygenase (15LOX) in sperm CD, we hypothesize that subfertile male Alox15 mice lacking the 15Lox gene display sperm CD anomalies. Caput and cauda epididymal sperm samples from seven adult Alox15 and seven wild-type (wt) males of equal age were examined by differential interference contrast microscopy (DIC) and transmission electron microscopy (TEM). Compared with wt males, Alox15 males had significantly more spermatozoa with a retained CD in both caput (P = 0.004) and cauda (P = 0.005) epididymidis. TEM and DIC analyses revealed intact mitochondria present in the CDs of epididymal Alox15 spermatozoa. The CDs of wt spermatozoa, however, had a smooth appearance and contained only hollow membrane vesicles, with no intact mitochondria embedded in their CD matrix. Epithelial lesions, phagocytosis-like figures, and missing or aberrant apical blebs were observed in the caput epididymidis of Alox15 males. Thus, the process of epididymal sperm maturation and CD migration is altered in Alox15 males. Aberrant sperm maturation might contribute to the reduced fertility and smaller litter size of Alox15 mice, a rare example of subfertile mutants displaying normal spermatogenesis but altered epididymal sperm maturation.

High resolution light microscopic evaluation of boar semen quality sperm cytoplasmic droplet retention in relationship with boar fertility parameters.

The purpose of this study was to investigate the relationship between fertility and quantitative measures of boar semen quality, including various patterns of sperm cytoplasmic droplet (CD) retention, as determined by high power differential interference contrast (DIC) microscopy. A total of 116 ejaculates were collected from a nucleus herd of 18 Large White boars over an eight month period. Semen quality parameters were analyzed for each ejaculate by calculating the percentage of normal spermatozoa, spermatozoa possessing a CD in the proximal, distal, or distal midpiece reflex position, total spermatozoa with an attached cytoplasmic droplet, spermatozoa with non-CD related aberrations and total spermatozoa with abnormalities. Of the 116 ejaculates received, 71 ejaculates from 13 boars had corresponding fertility data from single-sire inseminations of multiparous sows. The fertility data included farrowing rate (FR) and total number born (TNB). The monthly FR encompassed one month before and one month after the date of semen collection. Detection of differences for fertility and semen quality parameters was performed by separating the boars into either an above-average or below-average group based on the mean FR (74.01 ± 1.43%) or TNB (12.34 ± 0.17) for the study. For FR, the boars in the below-average group had a significantly lower percentage of normal spermatozoa and significantly higher percentage of spermatozoa possessing distal CDs, total attached CDs and total abnormalities compared to the boars in the above-average group. Conversely, for TNB there were no significant differences between the above- and below-average groups for the semen quality parameters. These data suggest that the attached CD may negatively affect FR, but not TNB. The detection of relationships between the boar fertility parameters and the retention of the sperm CD after ejaculation, document the advantage of high power DIC microscopy in conventional semen evaluation.