Influence of insemination time on pregnancy rates of mares inseminated with frozen semen
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collected from 10 stallions, aged from 10 to 23 years. Seminal plasma was removed by centrifugation and spermatozoa were washed by two successive centrifugations in Tyrode extender. Analysis was performed by immunocytochem- istry, western-blot, immunofluorescence and flow cytom- etry with MC-20 (ESR1), H-150 (ESR2) antibodies. GPER identification in equine species was confirmed by RT-PCR and sequencing. Both estrogen receptors ESR1 and ESR2 are present in stallion spermatozoa. For ESR1, western-blot analysis shows a single 66kDa band corresponding to the wild-type isoform; immunofluorescence analysis showed a flagellar staining. For ESR2, western-blot analysis shows a single 61 kDa band, corresponding to the molecular weight described for the wild type form of this receptor and confocal analysis demonstrated also a flagellar localization. The rate of spermatozoa positive for the detection of both of these receptors was analyzed by flow cytometry. 98% of spermatozoa were positive for ESR1 and 94% positive for ESR2 in samples from 3 stallions collected in April. The estrogen transmembrane receptor, GPER was also identified. Genome from Equus Caballus contains a GPER-like gene but identification and expression of this gene in horses has not been reported so far. We proceeded to extract RNA from a colt testis and spermatozoa from 3 stallions and with primers designed from the DNA sequence, RT-PCR amplifi- cation was performed. The products were sequenced and allowed to describe the expression of GPER in the equine species for the first time. In conclusion, the presence of estrogen receptors in stallion spermatozoa was demon- strated suggesting that sperm are a putative estrogen target. Moreover, GPER was described for the first time in horses. This is opening new possibilities to study estradiol action in male as well as in female reproduction. Effect of nitric oxide enzyme (NOS) inhibition and effect of nitric oxide (NO) scavenger on motility patterns and hyperactivated population of cryopreserved equine sperm R.P. Arruda 1 , D.F. Silva 1 , A.F.C. Andrade 1 , M.C. Caldas-Bussiere 2 , M.A. Alonso 1 , E.C.C. Celeghini 1 , K.M. Lemes 1 , H.F. Carvalho 1 , and S.A. Florez-Rodriguez 1 1 Laboratory of Semen Biotechnology and Andrology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil, 2 Laboratory of Animal Breeding and Reproduction, Center of Agriculture and Technology, State University of North Fluminense Darcy Ribeiro, Campos dos Goytacazes, Brazil The aim of this study was to determine the effect of NOS inhibition and effect of an NO scavenger on motility patterns and hyperactivated sperm in cryopreserved equine semen. Three ejaculates were collected from each of three stallions (n ¼ 9). Frozen-thawed semen was centrifuged in bovine in vitro fertilization (IVF) media supplemented with sodium bicarbonate and bovine serum albumin for sperm capacitation. The pellet was incubated in the same media with L-arginine, with or without the NOS inhibitor, Nu-nitro-L-arginine metylester (L-NAME) and in media with L-arginine with or without the NO scavenger, methylene blue: control T1¼ (C-IVF), T2¼ L-arginine 10mM, T3¼ L-NAME 1 mM, T4¼ methylene blue 100 mM, T5¼ L-arginine (10 mM) + L-NAME (1mM) and T6¼ L-arginine (10 mM) + methylene blue (100mM) for 0, 60, 120 and 300 minutes at 38 C (5% CO 2) . Sperm was considered hyperactivated when VCL 180mm/s and ALH 12 mm. Data were analyzed using ANOVA and subsequently compared by Tukey test (SAS). The presence of the scavenger in the culture media para- lysed equine sperm cells. Total motility, progressive motility and rapid cells differed among treatments at time 0 in groups T2 and T5 (P < 0.05), when compared with T1, T3, T4 and T6 at times 0, 60, 120 and 300 minutes. VAP was reduced (P < 0.05) when L-arginine was added. L-NAME did not reduce the effect of NO. At time 0, L-arginine addition reduced VSL (T2 51.8 2.8; T5 54.1 2.8) compared to groups T1 and T3 (T1 78.0 5.4; T3 77.2 5.6). VCL was not altered by any of the treatments in all the sampling times. There was no interaction between times and treatment for ALH. When comparing only treatment effect, groups T4 and T6 had a reduction in ALH compared to groups T1, T2, T3 and T5. Treatment effect on BCF only occurred after 120 minutes of incubation. At this time, BCF was lower in T1. There were no significant differences in STR when the treatments that do not cause sperm immobilization were analyzed. At time 0 LIN suffered a reduction in groups T2 and T5 (T2 32.3 1.9; T5 35.2 31.6). In group T5 a reduction (P < 0.05) in the percentage of hyperactivated cells was seen compared to groups T1, T2, T3, T4 and T6, at times 0, 120 and 300. Our results confirm that the presence of NO modulates sperm motility patterns although it does not induce hyperactivation when sperm are incubated with methylene blue. Acknowledgments The present study received financial support FAPESP (Grants 2009/54906-5). Influence of insemination time on pregnancy rates of mares inseminated with frozen semen B.R. Avanzi, R.S. Ramos, G.H.M. Araujo, E.G. Fioratti, G.M. Greco, L.A. Trinca, J.A. Dell'Aqua, Jr., C.M. Melo, and F.O. Papa Department of Animal Reproduction and Veterinary Radiology, UNESP Botucatu, SP, Brazil, Distrito de Rubião Junior S/N, 18618-970 One of the major limiting factors to the widespread use of frozen semen is the cost associated with the intense management of mares. It is generally recommended that frozen stallion semen should be inseminated within 12 h prior to ovulation and 6 h after ovulation. The aim of the present study was to compare pregnancy rates of mares inseminated 1) post-ovulation and 2) 40 h after ovulation induction. Semen was obtained from 2 stallions, A) a Westfalen of good fertility and B) a Mangalarga March- ador of poor fertility. Six ejaculates from each stallion were collected (n ¼ 12). The semen was diluted with Botu-Semen (1:1) and centrifuged at 600 x g for 10 minutes. The 6 th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518 476
Transcript of Influence of insemination time on pregnancy rates of mares inseminated with frozen semen
6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518476
collected from10 stallions, aged from10 to 23 years. Seminalplasma was removed by centrifugation and spermatozoawere washed by two successive centrifugations in Tyrodeextender. Analysis was performed by immunocytochem-istry, western-blot, immunofluorescence and flow cytom-etry with MC-20 (ESR1), H-150 (ESR2) antibodies. GPERidentification in equine species was confirmed by RT-PCRand sequencing. Both estrogen receptors ESR1 and ESR2 arepresent in stallion spermatozoa. For ESR1, western-blotanalysis shows a single 66kDa band corresponding to thewild-type isoform; immunofluorescence analysis showeda flagellar staining. For ESR2, western-blot analysis showsa single 61 kDa band, corresponding to themolecularweightdescribed for the wild type form of this receptor andconfocal analysis demonstrated also a flagellar localization.The rate of spermatozoa positive for the detection of both ofthese receptors was analyzed by flow cytometry. 98% ofspermatozoa were positive for ESR1 and 94% positive forESR2 in samples from 3 stallions collected in April. Theestrogen transmembrane receptor, GPERwas also identified.Genome from Equus Caballus contains a GPER-like gene butidentification and expression of this gene in horses has notbeen reported so far. We proceeded to extract RNA froma colt testis and spermatozoa from 3 stallions and withprimers designed from the DNA sequence, RT-PCR amplifi-cation was performed. The products were sequenced andallowed to describe the expression of GPER in the equinespecies for the first time. In conclusion, the presence ofestrogen receptors in stallion spermatozoa was demon-strated suggesting that sperm are a putative estrogen target.Moreover, GPER was described for the first time in horses.This is opening new possibilities to study estradiol action inmale as well as in female reproduction.
Effect of nitric oxide enzyme (NOS) inhibition and effectof nitric oxide (NO) scavenger on motility patterns andhyperactivated population of cryopreserved equinesperm
R.P. Arruda 1, D.F. Silva 1, A.F.C. Andrade 1,M.C. Caldas-Bussiere 2, M.A. Alonso 1,E.C.C. Celeghini 1, K.M. Lemes 1, H.F. Carvalho 1, andS.A. Florez-Rodriguez 1
1 Laboratory of Semen Biotechnology and Andrology,Department of Animal Reproduction, School of VeterinaryMedicine and Animal Science, University of São Paulo, SãoPaulo, Brazil, 2 Laboratory of Animal Breeding andReproduction, Center of Agriculture and Technology, StateUniversity of North Fluminense Darcy Ribeiro, Camposdos Goytacazes, Brazil
The aim of this study was to determine the effect of NOSinhibition and effect of an NO scavenger on motilitypatterns and hyperactivated sperm in cryopreserved equinesemen. Three ejaculates were collected from each of threestallions (n ¼ 9). Frozen-thawed semen was centrifuged inbovine in vitro fertilization (IVF) media supplemented withsodium bicarbonate and bovine serum albumin for spermcapacitation. The pellet was incubated in the same mediawith L-arginine, with or without the NOS inhibitor,Nu-nitro-L-argininemetylester (L-NAME) and inmediawith
L-argininewith orwithout theNO scavenger,methylene blue:control T1¼ (C-IVF), T2¼ L-arginine 10mM, T3¼ L-NAME 1mM, T4¼methylene blue 100mM, T5¼ L-arginine (10mM)+ L-NAME (1mM) and T6¼ L-arginine (10 mM) +methyleneblue (100mM) for 0, 60, 120 and 300 minutes at 38�C (5%CO2). Sperm was considered hyperactivated when VCL�180mm/s and ALH �12 mm. Data were analyzed usingANOVA and subsequently compared by Tukey test (SAS).The presence of the scavenger in the culture media para-lysed equine sperm cells. Totalmotility, progressivemotilityand rapid cells differed among treatments at time 0 ingroups T2 and T5 (P< 0.05), when comparedwith T1, T3, T4and T6 at times 0, 60, 120 and 300 minutes. VAP wasreduced (P< 0.05) when L-argininewas added. L-NAME didnot reduce the effect of NO. At time 0, L-arginine additionreduced VSL (T2 51.8 � 2.8; T5 54.1 � 2.8) compared togroups T1 and T3 (T178.0� 5.4; T3 77.2� 5.6). VCL was notaltered by any of the treatments in all the sampling times.There was no interaction between times and treatment forALH.When comparing only treatment effect, groups T4 andT6 had a reduction in ALH compared to groups T1, T2, T3and T5. Treatment effect on BCF only occurred after 120minutes of incubation. At this time, BCF was lower in T1.There were no significant differences in STR when thetreatments that do not cause sperm immobilization wereanalyzed. At time 0 LIN suffered a reduction in groups T2and T5 (T2 32.3 � 1.9; T5 35.2 � 31.6). In group T5a reduction (P < 0.05) in the percentage of hyperactivatedcells was seen compared to groups T1, T2, T3, T4 and T6, attimes 0, 120 and 300. Our results confirm that the presenceof NO modulates sperm motility patterns although it doesnot induce hyperactivationwhen sperm are incubated withmethylene blue.
Acknowledgments
The present study received financial support FAPESP(Grants 2009/54906-5).
Influence of insemination time on pregnancy rates ofmares inseminated with frozen semen
B.R. Avanzi, R.S. Ramos, G.H.M. Araujo, E.G. Fioratti,G.M. Greco, L.A. Trinca, J.A. Dell'Aqua, Jr., C.M. Melo, andF.O. PapaDepartment of Animal Reproduction and VeterinaryRadiology, UNESP Botucatu, SP, Brazil, Distrito de RubiãoJunior S/N, 18618-970
One of the major limiting factors to the widespread use offrozen semen is the cost associated with the intensemanagement of mares. It is generally recommended thatfrozen stallion semen should be inseminated within 12 hprior to ovulation and 6 h after ovulation. The aim of thepresent study was to compare pregnancy rates of maresinseminated 1) post-ovulation and 2) 40 h after ovulationinduction. Semen was obtained from 2 stallions, A)a Westfalen of good fertility and B) a Mangalarga March-ador of poor fertility. Six ejaculates from each stallion werecollected (n¼ 12). The semenwas dilutedwith Botu-Semen(1:1) and centrifuged at 600 x g for 10 minutes. The
6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518 477
supernatant was discarded and the pellet resuspended inBotucrio extender. Semen was packed into 0.5mL strawsthat were kept at 5oC for 20 minutes. The straws were thenplaced 6 cm above liquid nitrogen level for 20 minutes tofreeze and finally plunged into it. Straws were thawed at46�C for 20 seconds. The insemination dose was 400x106
total sperm. The samples were analyzed by CASA (HTM –
IVOS 12), and plasmamembrane integrity was evaluated byfluorescent stains (CFDA and PI). The fertility trial wascarried out with 29 mares: 15 were inseminated withstallion A (30 cycles) and 14 with stallion B (28 cycles). Theestrus of the mares was detected via transrectal palpationand ultrasonography. One milligram of deslorelin acetatewas administered intramuscularly when a preovulatoryfollicle of at least 35 mm in diameter was detected. Eigh-teen hours after the induction of ovulation, the mares werepalpated every 6 hours until ovulation was detected. Forprotocol (1), the mares were inseminated no more than 6hours after ovulation. For protocol (2), the mares wereinseminated 40 hours after the deslorelin acetate injection.All mares were inseminated into the tip of the ipsilateraluterine horn. Semen data were analyzed by ANOVA usingProc GLM. Pregnancy rates among stallions and insemina-tion procedures were compared by logistic regressionanalysis using Proc GENMOD. Significance was set as P <
0.05. Values for total motility (77.3 � 3.4a and 70.5 � 1.3b),progressive motility (42.1 � 3.1a and 27.0 � 1.4b), % rapidcells (65.3 � 4.0a and 47.0 � 2.9b) and plasma membraneintegrity (34.0 � 3.1 and 31.7 � 4.8) were obtained forstallion A and B, respectively (different letters showsignificant statistical difference). Pregnancy rates were46.7% (7/15) and 35.7% (5/14) when breed with 6h afterovulation with semen from stallions A and B, respectively.As for inseminations performed 40h after ovulationinduction, pregnancy rates were 66.6% (10/15) and 21.4%(3/14) for stallions A and B, respectively. Based on theresults of the present study we can conclude that insemi-nation at a fixed time after ovulation induction usingequine frozen semen was efficient. Thus, the present datasupports the feasibility of equine frozen semen as a tech-nology to be widely used and spread since it reduces maremanagement and costs.
Acknowledgments
Financial support: FAPESP.
Effect of sex sorting on intracellular Ca2+ and ATPcontent of stallion spermatozoa
C. Balao-da Silva, A. Morillo-Rodriguez,C. Ortega-Ferrusola, J.M. Gallardo-Bolaños,M. Plaza-Dávila, A. Miro-Morán, I.M. Aparicio,J.A. Tapia, and F.J. PeñaFaculty of Veterinary Medicine, University ofExtremadura, Cáceres, Spain
Sex sorting flow cytometry allows the selection of X and/or Y chromosome bearing spermatozoa. This procedure
may damage sperm through the effects of high pressureand dilution, U.V light exposure, effect of the dye andmechanical forces. Thus, understanding the mechanismsbehind the damage is required, in order to improve thetechnique in horses. So far, the effects of sorting on theenergy status and calcium homeostasis have not beenaddressed. Several sperm functions require substantialamounts of ATP, such as flagellar movement and signaltransduction via protein phosphorylation. Energy can beproduced by oxidative phosphorylation at the mito-chondria, but also by glycolysis. Calcium status has beendirectly related to flagellar modulation, and thereby,spermatic motility. Five pools of semen from threedifferent stallions were made. At first, selection ofspermatozoa was made through the use of a colloid witha species-specific formulation (Androcoll-E, Uppsala,Sweden). Afterwards, the sample was diluted in INRA-Tyrodes, stained with Hoechst 33344 and incubated at34oC. Spermatozoa were sorted in a MoFLo SX DP(Beckman Coulter, Miami, FL, USA). Kinematic parame-ters were estimated by CASA. Evaluation of spermatozoalstatus was assessed by flow cytometry: viability with thecombination of probes Yo-Pro-1 and Ethidium Homo-dimer; mitochondrial membrane potential with JC-1probe and Calcium detection with Fluo3. A biolumines-cence assay (ATP Determination Kit A22066) was alsoperformed, to quantify the amount of ATP present. Allreagents were from Molecular Probes (Leiden, TheNetherlands). After sex-sorting, total and progressivemotility decreased significantly, while late apoptotic anddead spermatozoa subpopulations increased. Live cellsalso decreased significantly. This occurred in parallel toa slight decrease in the intracellular percentage ofCa2+ in living cells of non-sorted (44.9 � 9.51) andsorted (40.7 � 9.51) samples. Regarding ATP detection,a decrease of approximately 40% between the pre-sortedand sorted sample was detected (measured in pM/100mgprotein). Association of ATP levels with sperm kinematicparameters (already made in our laboratory) arecorroborated by this study, since both ATP and motilityvalues decreased. As mitochondrial membrane potentialwas maintained, we could relate this parameter withother cell maintenance processes, rather than flagellarmodulation. On the other hand, ATP could still beproduced by oxidative phosphorylation, and this loss canbe explained by the opening of some membrane pores.This might be coincident with the increase in lateapoptotic spermatozoa, and therefore, plasma membranepermeabilization. In summary, the intracellular ATPdecrease in stallion spermatozoa is related to kinematicparameters such as total and progressive motility. Also,apoptotic and dead cells are associated with a decreasein ATP content. Calcium may also play a role on thiskinematic decrease.
Acknowledgments
Funded by: AGL 2010-20758 (GAN).
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