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134 l -Carnitine and pyruvate inclusion in diluents for cold-stored stallion spermatozoa
440
Abstracts / Journal of Equine Veterinary Science 35 (2015) 437e445
Testicular tissue was collected from 6 pre-pubertal (333 ± 49 d) and 13 post-pubertal (1,480 ± 396 d) stallions during routine castrations at the Kansas State University Veterinary Medical School. Total RNA extracted from testicular tissue (n ¼ 11), was reverse transcribed and mRNA for GnRH-I, GnRHR-I, GnRH-II, and GnRHR-II were examined using gene specific primers and conventional PCR. The identity of the PCR products was confirmed via sequencing at the University of Nebraska Medical Center. Gel electrophoresis of PCR products identified the presence of both ligand and receptor isoforms in pre- and postpubertal stallions (n ¼ 11). Protein was extracted from testicular tissue and subjected to immunoblotting using antibodies directed against either GnRHR-I (sc-8681; Santa Cruz Biotechnology) or GnRHR-II (sc-100301; Santa Cruz Biotechnology). Western blot results demonstrated that testicular samples from pre-pubertal stallions (n ¼ 4) had 5-fold greater GnRHR-I levels compared with post-pubertal animals (n ¼ 7; P < 0.0005). Conversely, amounts of GnRHR-II protein were 3.3-fold greater in post-pubertal animals (n ¼ 10) compared with pre-pubertal stallions (n ¼ 4; P < 0.0001). The presence of GnRH isoforms and their receptors in the equine testis suggests a possible autocrine or paracrine function.
Key Words: GnRH-I, GnRH-II, equine
133 Differential miRNA expression from sperm obtained in the different regions of the stallion epididymis H.M. Twenter*, K.M. Klohonatz, A.D. Belk, L.D. Bass, G.J. Bouma, and J.E. Bruemmer Colorado State University, Fort Collins, CO, USA Following spermatogenesis, spermatozoa travel through the convoluted tubules that comprise the epididymis. As sperm cells migrate through the epididymis they undergo various changes in the different regions. Sperm enter at the caput where fluid is absorbed to change the concentration of the sperm. As the cells travel through the corpus of the epididymis they undergo maturation including migration and ultimate loss of the cytoplasmic droplet. Once the cells reach the cauda of the epididymis, they will be stored until ejaculation or removed through urination. We hypothesize that as sperm travel through the epididymis they not only undergo changes to fertilize an oocyte, but also changes to their miRNA content. Our objective was to evaluate sperm from the cauda and caput of the epididymis to compare the miRNA expression. Epididymides were collected from 3 mature stallions following elective routine surgical castration and segmented into 4 regions: the cauda, proximal corpus, distal corpus, and the caput. The tissue was then placed in modified Whitten’s medium, punctured, and centrifuged to extract sperm cells from each section. Sperm cells were collected from the medium with Equipure (Nidaco) and resuspended in phosphate-buffered saline. Total RNA was extracted and used for quantitative real time PCR. Samples were evaluated for the presence and quantity of 346 equine specific miRNAs. A Student’s t-test was used to compare the expression levels, with P < 0.05 indicating significant differential expression. Ten miRNAs were differentially expressed, all of which were upregulated in the caudal sperm as compared with those isolated in the caput. Although the specific functions of these miRNA have not yet been determined, they may play a role in final maturation or storage of sperm. Two miRNAs were also found solely in the caudal samples while 6 were only found in the less mature sperm of the caput. Better understanding of the roles of these
miRNA may lead to new management options with stallions facing fertility issues.
Key Words: sperm, miRNA, epididymis 134 L-Carnitine
and pyruvate inclusion in diluents for cold-stored stallion spermatozoa D.S. Potter*, G.W. Webb, B. Onyango, and T. Northcutt Missouri State University, Springfield, MO, USA L-Carnitine (CARN), a compound created from lysine and methionine, is produced within the body of most animals. Carnitine has been studied for its effectiveness in energy production, specifically relating to its role in the mobilization of fatty acids for ATP. Recently, inclusion of CARN and pyruvate has been reported to improve post-storage motility of stallion spermatozoa. Two experiments were conducted with the objective of determining if addition of CARN and pyruvate (CARN+P) to 3 commonly used semen extenders would improve the motility and viability of cold-stored stallion spermatozoa. Total (TM) and progressive motility (PM), velocity and direction of movement (VAP, VSL, VCL, and elongation) were analyzed by computer assisted sperm analysis (CASA). Flow-cytometry was used to assess membrane status with SYBR 14 and propidium iodide (PI) stains and acrosome status using the FITC-PSA stain. In experiment 1 (EX1) 4 ejaculates were collected from each of 2 stallions. Aliquots of each ejaculate were diluted in either Kenney’s skimmilk glucose extender (SKMG) or INRA 96 (INRA) with and without CARN and pyruvate added at levels of 0.00806 g/mL and 0.0011004 g/mL for a total of 4 extender treatments. Split aliquots of each treatment were placed in separate Equine Express II disposable containers for CASA after 2 h, 24 h, and 48 h of storage. Flow-cytometeric analysis was conducted only at 24 h. In experiment 2 (EX2), 4 ejaculates were collected from each of 3 stallions. Aliquots of each were diluted in SKMG, INRA, and Revolution (REV) with or without CARN+P (6 extender treatments). Data were analyzed using the GLM function of ANOVA. Pairwise comparisons were made with Tukey’s test. Data were analyzed across all stallions/ejaculates/times for effect of CARN+P and extender. Ejaculate was nested within stallion and time nested within ejaculate. In both experiments dilution in INRA resulted in higher motility compared with dilution in other extenders (P < 0.05). In EX1 CARN+P (P < 0.01) improved TM, PM, VAP, VCL, elongation. Intact membrane percentages were higher (P < 0.05) for spermatozoa diluted with CARN + P (74.32% with vs. 71.68% without). In EX2 CARN+P (P < 0.05) improved TM, PM, VAP, VSL, VCL and also improved (P < 0.05) the percentage of intact membranes (78.02% with vs. 75.03% without) and intact acrosomes (78.85% with vs. 66.22% without). In conclusion, addition of L-carnitine + pyruvate to extenders improved motility, velocity of movement and percentage of intact membranes and acrosomes of cold-stored stallion spermatozoa in these experiments.
Key Words: stallion, spermatozoa, carnitine
135 Effect of hydrogen peroxide and heat stress on stallion sperm function A.M. Mesa*, R.L. Roberson, R.I. Chun, and C.J. Mortensen University of Florida, Gainesville, FL, USA Spermatozoa are highly specialized cells, sensitive to environmental factors such as temperature and redox state. Increased
Abstracts / Journal of Equine Veterinary Science 35 (2015) 437e445
Testicular tissue was collected from 6 pre-pubertal (333 ± 49 d) and 13 post-pubertal (1,480 ± 396 d) stallions during routine castrations at the Kansas State University Veterinary Medical School. Total RNA extracted from testicular tissue (n ¼ 11), was reverse transcribed and mRNA for GnRH-I, GnRHR-I, GnRH-II, and GnRHR-II were examined using gene specific primers and conventional PCR. The identity of the PCR products was confirmed via sequencing at the University of Nebraska Medical Center. Gel electrophoresis of PCR products identified the presence of both ligand and receptor isoforms in pre- and postpubertal stallions (n ¼ 11). Protein was extracted from testicular tissue and subjected to immunoblotting using antibodies directed against either GnRHR-I (sc-8681; Santa Cruz Biotechnology) or GnRHR-II (sc-100301; Santa Cruz Biotechnology). Western blot results demonstrated that testicular samples from pre-pubertal stallions (n ¼ 4) had 5-fold greater GnRHR-I levels compared with post-pubertal animals (n ¼ 7; P < 0.0005). Conversely, amounts of GnRHR-II protein were 3.3-fold greater in post-pubertal animals (n ¼ 10) compared with pre-pubertal stallions (n ¼ 4; P < 0.0001). The presence of GnRH isoforms and their receptors in the equine testis suggests a possible autocrine or paracrine function.
Key Words: GnRH-I, GnRH-II, equine
133 Differential miRNA expression from sperm obtained in the different regions of the stallion epididymis H.M. Twenter*, K.M. Klohonatz, A.D. Belk, L.D. Bass, G.J. Bouma, and J.E. Bruemmer Colorado State University, Fort Collins, CO, USA Following spermatogenesis, spermatozoa travel through the convoluted tubules that comprise the epididymis. As sperm cells migrate through the epididymis they undergo various changes in the different regions. Sperm enter at the caput where fluid is absorbed to change the concentration of the sperm. As the cells travel through the corpus of the epididymis they undergo maturation including migration and ultimate loss of the cytoplasmic droplet. Once the cells reach the cauda of the epididymis, they will be stored until ejaculation or removed through urination. We hypothesize that as sperm travel through the epididymis they not only undergo changes to fertilize an oocyte, but also changes to their miRNA content. Our objective was to evaluate sperm from the cauda and caput of the epididymis to compare the miRNA expression. Epididymides were collected from 3 mature stallions following elective routine surgical castration and segmented into 4 regions: the cauda, proximal corpus, distal corpus, and the caput. The tissue was then placed in modified Whitten’s medium, punctured, and centrifuged to extract sperm cells from each section. Sperm cells were collected from the medium with Equipure (Nidaco) and resuspended in phosphate-buffered saline. Total RNA was extracted and used for quantitative real time PCR. Samples were evaluated for the presence and quantity of 346 equine specific miRNAs. A Student’s t-test was used to compare the expression levels, with P < 0.05 indicating significant differential expression. Ten miRNAs were differentially expressed, all of which were upregulated in the caudal sperm as compared with those isolated in the caput. Although the specific functions of these miRNA have not yet been determined, they may play a role in final maturation or storage of sperm. Two miRNAs were also found solely in the caudal samples while 6 were only found in the less mature sperm of the caput. Better understanding of the roles of these
miRNA may lead to new management options with stallions facing fertility issues.
Key Words: sperm, miRNA, epididymis 134 L-Carnitine
and pyruvate inclusion in diluents for cold-stored stallion spermatozoa D.S. Potter*, G.W. Webb, B. Onyango, and T. Northcutt Missouri State University, Springfield, MO, USA L-Carnitine (CARN), a compound created from lysine and methionine, is produced within the body of most animals. Carnitine has been studied for its effectiveness in energy production, specifically relating to its role in the mobilization of fatty acids for ATP. Recently, inclusion of CARN and pyruvate has been reported to improve post-storage motility of stallion spermatozoa. Two experiments were conducted with the objective of determining if addition of CARN and pyruvate (CARN+P) to 3 commonly used semen extenders would improve the motility and viability of cold-stored stallion spermatozoa. Total (TM) and progressive motility (PM), velocity and direction of movement (VAP, VSL, VCL, and elongation) were analyzed by computer assisted sperm analysis (CASA). Flow-cytometry was used to assess membrane status with SYBR 14 and propidium iodide (PI) stains and acrosome status using the FITC-PSA stain. In experiment 1 (EX1) 4 ejaculates were collected from each of 2 stallions. Aliquots of each ejaculate were diluted in either Kenney’s skimmilk glucose extender (SKMG) or INRA 96 (INRA) with and without CARN and pyruvate added at levels of 0.00806 g/mL and 0.0011004 g/mL for a total of 4 extender treatments. Split aliquots of each treatment were placed in separate Equine Express II disposable containers for CASA after 2 h, 24 h, and 48 h of storage. Flow-cytometeric analysis was conducted only at 24 h. In experiment 2 (EX2), 4 ejaculates were collected from each of 3 stallions. Aliquots of each were diluted in SKMG, INRA, and Revolution (REV) with or without CARN+P (6 extender treatments). Data were analyzed using the GLM function of ANOVA. Pairwise comparisons were made with Tukey’s test. Data were analyzed across all stallions/ejaculates/times for effect of CARN+P and extender. Ejaculate was nested within stallion and time nested within ejaculate. In both experiments dilution in INRA resulted in higher motility compared with dilution in other extenders (P < 0.05). In EX1 CARN+P (P < 0.01) improved TM, PM, VAP, VCL, elongation. Intact membrane percentages were higher (P < 0.05) for spermatozoa diluted with CARN + P (74.32% with vs. 71.68% without). In EX2 CARN+P (P < 0.05) improved TM, PM, VAP, VSL, VCL and also improved (P < 0.05) the percentage of intact membranes (78.02% with vs. 75.03% without) and intact acrosomes (78.85% with vs. 66.22% without). In conclusion, addition of L-carnitine + pyruvate to extenders improved motility, velocity of movement and percentage of intact membranes and acrosomes of cold-stored stallion spermatozoa in these experiments.
Key Words: stallion, spermatozoa, carnitine
135 Effect of hydrogen peroxide and heat stress on stallion sperm function A.M. Mesa*, R.L. Roberson, R.I. Chun, and C.J. Mortensen University of Florida, Gainesville, FL, USA Spermatozoa are highly specialized cells, sensitive to environmental factors such as temperature and redox state. Increased