M. A. Crenshaw

The use of digital infrared thermal imaging to detect estrus in gilts

Yorkshire/Landrace crossbred gilts (N = 32) were evaluated using digital infrared thermal imaging (DITI) to discriminate between estrus and diestrus phases of the porcine estrous cycle. Gilts (N = 32) were part of an ongoing reproductive efficiency study involving the use of raw soybean (RSB; N = 15) versus soybean meal (SBM; N = 17) as a source of dietary protein. Gilts were monitored daily for signs of estrus using a teaser boar. Thermal images of vulva surface temperatures (TEMP) were recorded at standing estrus and diestrus. Measurements for analysis included minimum (MIN), maximum (MAX), mean (AVG), and standard deviation (SD) of temperature gradients. At imaging, ambient (AMB) and rectal temperatures (RT) were recorded, and blood samples taken for serum progesterone (P(4)) concentration analysis (by RIA) to confirm stage of cycle. Mean serum progesterone values at estrus and diestrus were (mean ± SD) 1.0 ± 0.1 and 10.9 ± 0.8 ng/mL, respectively. Vulva MIN, MAX, and AVG thermal images were positively correlated with one another (P < 0.01), and were positively correlated with ambient temperature (P < 0.01). Vulva MAX and AVG thermal temperatures were greater (P < 0.05) at estrus than at diestrus (36.6 ± 0.2 °C and 33.4 ± 0.3 °C vs. 35.6 ± 0.3 °C and 31.8 ± 0.6 °C, respectively), whereas MIN and SD had no differences (P > 0.05) between stages of the cycle. No differences (P > 0.05) in RT were detected between stages and RT was not significantly correlated with vulva thermal images. Diet had no significant effect on RT or vulva temperature.

Lectin-Functionalized Magnetic Iron Oxide Nanoparticles for Reproductive Improvement

Background: Semen ejaculates contain heterogeneous sperm populations that can jeopardize male fertility. Recent development of nanotechnology in physiological systems may have applications in reproductive biology. Here, we used magnetic nanoparticles as a novel strategy for sperm purification to improve semen fertility. Methods: Boar semen was obtained in artificial insemination doses from a local boar stud. Doses were mixed with or without magnetic nanoparticles designed to target and deplete moribund and poor performing spermatozoa under an electromagnetic field. Sperm motility characteristics were assessed prior to insemination of open gilts with control (n=3 gilts) and nanopurified (n=4 gilts) semen. Pregnancies were verified 30 days post-insemination. Litter sizes and post-natal development of piglets were respectively evaluated at parturition and weekly until weaning. Results: Nanopurification significantly improved sperm motility. Two gilts in the control group were confirmed nonpregnant, but the remainder maintained pregnancies through to parturition (33% vs. 100%, control and nanopurified groups, respectively). At parturition, the number of piglets born to the control gilt was not significantly different from the average of the nanopurified group (17 ± 0.0 vs. 15 ± 2, respectively; P>0.05); however, in the latter group 78% of piglets remained alive compared to 76% of the control. Birth weight of control piglets was lower (1.18 ± 0.22 kg) than those in the nanopurified group (1.41 ± 0.14 kg). Both groups of piglets showed linear and parallel growth rates with respective weight gains of 4.4x and 4.1x from birth to weaning. Interestingly, piglets produced in the nanopurified group comprised of 55% males compared to 38% in the control group. Conclusions: Magnetic nanoparticles used in this preliminary study exhibited no toxic effects on sperm fertilization capacity and piglet viability. Beneficial effects may be seen in semen fertility, with possible use for gender selection. Further investigations on a large scale are needed to confirm the current findings, with potential application in clinical practice.

Profiling of relaxin and its receptor proteins in boar reproductive tissues and spermatozoa

BackgroundRelaxin levels in seminal plasma have been associated with positive effects on sperm motility and quality, and thus having potential roles in male fertility. However, the origin of seminal relaxin, within the male reproductive tract, and the moment of its release in the vicinity of spermatozoa remain unclear. Here, we assessed the longitudinal distribution of relaxin and its receptors RXFP1 and RXFP2 in the reproductive tract, sex accessory glands, and spermatozoa of adult boars.MethodsSpermatozoa were harvested from three fertile boars and reproductive tract (testes and epididymis) and sex accessory gland (prostate and seminal vesicles) tissues were collected post-mortem from each boar. Epididymis ducts were sectioned into caput, corpus, and cauda regions, and spermatozoa were mechanically collected. All samples were subjected to immunofluorescence and/or western immunoblotting for relaxin, RXFP1, and RXFP2 detection. Immunolabeled-spermatozoa were submitted to flow cytometry analyses and data were statistically analyzed with ANOVA.ResultsBoth receptors were detected in all tissues, with a predominance of mature and immature isoforms of RXFP1 and RXFP2, respectively. Relaxin signals were found in the testes, with Leydig cells displaying the highest intensity compared to other testicular cells. The testicular immunofluorescence intensity of relaxin was greater than that of other tissues. Epithelial basal cells exhibited the highest relaxin immunofluorescence intensity within the epididymis and the vas deferens. The luminal immunoreactivity to relaxin was detected in the seminiferous tubule, epididymis, and vas deferens ducts. Epididymal and ejaculated spermatozoa were immunopositive to relaxin, RXFP1, and RXFP2, and epididymal corpus-derived spermatozoa had the highest immunoreactivities across epididymal sections. Both vas deferens-collected and ejaculated spermatozoa displayed comparable, but lowest immunofluorescence signals among groups. The entire sperm length was immunopositive to both relaxin and receptors, with relaxin signal being robust in the acrosome area and RXFP2, homogeneously distributed than RXFP1 on the head of ejaculated spermatozoa.ConclusionsImmunolocalization indicates that relaxin-receptor complexes may have important roles in boar reproduction and that spermatozoa are already exposed to relaxin upon their production. The findings suggest autocrine and/or paracrine actions of relaxin on spermatozoa, either before or after ejaculation, which have possible roles on the fertilizing potential of spermatozoa.

68 GROWTH AND MARKET QUALITY OF PIGS BORN FROM MAGNETIC NANOPARTICLE-TREATED BOAR SPERMATOZOA

Sperm ejaculates contain a heterogeneous population of nonviable and viable spermatozoal cells. Ejaculates with high concentrations of poor quality or damaged spermatozoa can greatly impair the overall fertility of males. Recently, a novel technique termed nanopurification has been developed (Feugang et al. 2015 IVF Reprod. Med. Genet. 3, 2) to noninvasively target and remove poor quality spermatozoa from boar semen. Such removal will enrich insemination doses with high quality spermatozoa to enhance fertility successes. However, effects associated with offspring born from nanopurified semen and possible meat quality assurance have yet to be extensively studied. The objective of this study was to measure the growth performance and market characteristics of pigs born from standard or nanopurified spermatozoa. Boar semen was obtained in insemination doses from a local stud and was mixed with (nanopurified) magnetic nanoparticles (iron-oxide) specifically designed to interact with acrosome-reacted and apoptotic spermatozoa. After incubation, mixed semen were placed under an electromagnetic field trapping moribund sperm to allow collection of intact and viable spermatozoa. Six gilts were bred with standard non-purified (control; n=3) or nanopurified (n=3) semen, with subsequent pregnancies leading to full-term birth of viable offspring. At weaning, pigs of equal sexes (5 male and 5 female) were randomly selected from control (n=10) and nanopurified (n=10) litters. Pigs were fed and measured until market weight, at which meat quality and carcass characteristics were assessed. Data (mean±SEM) were analysed with Students t-test and SAS software (SAS Institute Inc., Cary, NC, USA). The threshold of significance was set as P<0.05. Patterns of growth between groups were comparable up to market size (P>0.05). Standard pork quality parameters (lean carcass weight, loin eye area, percentage of lean cuts, loin and ham colouring, etc.) revealed no significant differences between groups (P>0.05). Dressing percentage was found higher in the nanopurified group compared with control, with a 1.5% increase (P<0.05). Marbling score tended to be significantly higher in the nanopurified group (2.7±0.15) when compared with the control (2.3±0.15). Findings indicate that sperm nanopurification does not impair growth of offspring and could ultimately lead to a higher pork carcass quality. Additional research is being conducted to confirm current findings and identify further effects of nanopurification regarding offspring and carcass quality.

A Systems Biology Approach Using Transcriptomic Data Reveals Genes and Pathways in Porcine Skeletal Muscle Affected by Dietary Lysine

Nine crossbred finishing barrows (body weight 94.4 ± 6.7 kg) randomly assigned to three dietary treatments were used to investigate the effects of dietary lysine on muscle growth related metabolic and signaling pathways. Muscle samples were collected from the longissimus dorsi of individual pigs after feeding the lysine-deficient (4.30 g/kg), lysine-adequate (7.10 g/kg), or lysine-excess (9.80 g/kg) diet for five weeks, and the total RNA was extracted afterwards. Affymetrix Porcine Gene 1.0 ST Array was used to quantify the expression levels of 19,211 genes. Statistical ANOVA analysis of the microarray data showed that 674 transcripts were differentially expressed (at p ≤ 0.05 level); 60 out of 131 transcripts (at p ≤ 0.01 level) were annotated in the NetAffx database. Ingenuity pathway analysis showed that dietary lysine deficiency may lead to: (1) increased muscle protein degradation via the ubiquitination pathway as indicated by the up-regulated DNAJA1, HSP90AB1 and UBE2B mRNA; (2) reduced muscle protein synthesis via the up-regulated RND3 and ZIC1 mRNA; (3) increased serine and glycine synthesis via the up-regulated PHGDH and PSPH mRNA; and (4) increased lipid accumulation via the up-regulated ME1, SCD, and CIDEC mRNA. Dietary lysine excess may lead to: (1) decreased muscle protein degradation via the down-regulated DNAJA1, HSP90AA1, HSPH1, and UBE2D3 mRNA; and (2) reduced lipid biosynthesis via the down-regulated CFD and ME1 mRNA. Collectively, dietary lysine may function as a signaling molecule to regulate protein turnover and lipid metabolism in the skeletal muscle of finishing pigs.

Effects of dietary lysine level on the content and fatty acid composition of intramuscular fat in late-stage finishing pigs

Abstract: This study was conducted to investigate how dietary lysine level affects the intramuscular fat (IMF) content and fatty acid (FA) composition in late-stage finishing pigs. Nine crossbred barrows [94.4 ± 6.7 kg body weight (BW)] were randomly allotted to three treatment groups (n = 3). Three corn- and soybean-meal-based diets were formulated to meet the National Research Council (2012) requirements for various nutrients except for lysine, whose concentrations were 0.43%, 0.71%, and 0.98% (as-fed basis) for Diets 1 (lysine-deficient), 2 (lysine-adequate), and 3 (lysine-excess), respectively. After 5 wk of ad libitum access to diets, pigs were harvested and longissimus dorsi samples were collected. The IMF content and FA composition of the samples were analyzed by gas chromatography. Results showed that the IMF content of the muscle was increased linearly (P < 0.05) with decreasing dietary lysine level from 0.98% to 0.43%. Dietary lysine level altered the composition of FA, especially the unsaturated FA, in the muscle. Particularly, the percentages of C18:1 n-9 and total monounsaturated FA were higher, whereas the percentages of C18:2 n-6 and total polyunsaturated FA were lower, in the muscle of the pigs fed Diet 1. Collectively, dietary lysine deficiency increased the proportion of monounsaturated FA and decreased the proportion of polyunsaturated FA, which may benefit pork palatability.

Effects of dietary supplementation of l-methionine vs. dl-methionine on performance, plasma concentrations of free amino acids and other metabolites, and myogenesis gene expression in young growing pigs

Abstract Methionine (Met), the second or third limiting amino acid (AA) in typical swine diets, plays important roles in promoting swine health and growth, especially, muscle growth. Whereas dl-Met products have been used in swine industry for many years, l-Met products have been developed recently. This research was conducted to study the effects of supplemental l-Met or dl-Met on nutrient metabolism, muscle gene expression, and growth performance of pigs. Twenty crossbred young barrows (initial body weight [BW] 21.2 ± 2.7 kg) were randomly assigned to 20 individual pens and two dietary treatments according to a completely randomized design with pigs serving as the experiment unit (n = 10). Two corn and soybean meal-based diets (diets 1 and 2) were formulated to meet or exceed the recommended requirements for energy, AA, and other nutrients (NRC. 2012. Nutrient requirements of swine, 11th ed. Washington, DC: The National Academies Press; AMINODat 5.0). Crystalline l-Met and dl-Met were supplemented to diets 1 and 2 (both at 0.13%, as-fed basis), respectively. After 4 wk of an ad libitum feeding trial, BW and feed intake were measured to calculate average daily gain (ADG), average daily feed intake (ADFI), and gain-to-feed ratio (G:F). Blood samples were collected from the jugular vein for analyses of plasma AA and metabolite concentrations. The longissimus dorsi muscle samples were collected for analysis of myogenesis gene expression. Data were analyzed using Student’s t-test. There were no differences (P = 0.56 to 0.94) in ADG, ADFI, or G:F between pigs fed the two experimental diets and no differences between diets were observed in plasma free AA concentrations. No differences were observed between pigs fed the two diets in expression of mRNA for eight myogenesis-related genes, which were myogenic differentiation 1, myogenin, myogenic factors 5, muscle regulatory factor 4 (a.k.a. myogenic factors 6), and myocyte enhancer factors 2A, 2B, 2C, and 2D. In conclusion, results of this experiment indicate that the bioefficacy of l-Met is not different from that of dl-Met, which is likely because of an efficient conversion of d-Met to l-Met by pigs.