Equine seminal plasma and sperm membrane: Functional proteomic assessment.

During ejaculation, a large amount of seminal plasma proteins interact with the sperm membrane, leading to a series of biochemical and structural changes implicated in sperm function and gamete interaction. However, the roles of the majority of these proteins remain unknown. This study aimed to investigate the proteome and functionality of the major equine proteins of seminal plasma and the sperm membrane. Seminal plasma and enriched-membrane proteins (150 µg) were separated by two-dimensional gel electrophoresis, and the respective maps were analyzed. Protein identification was performed by in-gel digestion and tandem mass spectrometry (GeLC-MS/MS). Samples were also submitted to in-solution digestion (complex protein mixture) and identified by shotgun analysis by LC-MS/MS; bioinformatic tools were used to investigate protein functions. Seminal plasma and sperm membrane extract maps contained 91.0 ± 8.2 spots and 245.3 ± 11.3 spots, respectively, within the 3-10 pH range. In total, the most abundant proteins identified in 2D maps and in complex protein mixtures included 24 proteins for seminal plasma and 33 for sperm membrane extract, with a high degree of confidence (P < 0.05). Of these, HSP1, CRISP3 and KLK1E2 were the most abundant in seminal plasma; HSP1 was highly abundant in sperm membrane extract, in many isoforms, which is related to membrane destabilization and may compromise sperm preservation. HSP1-polybromo-1 interactions suggested a role in DNA stabilization. Prosaposin was identified in seminal plasma and may play a role in the fertilization process. IZUMO4, a member of the IgSF family involved in the prefertilization stages, was identified in 2D gel and MS/MS analysis of sperm membrane extract. Ten proteins of seminal plasma were found to interact with the sperm membrane and were related to binding and catalytic activities (clusterin, CRISP3, epididymal sperm-binding protein 1, kallikrein1E2, seminal plasma protein A3, and HSP1). Additionally, other identified proteins were associated with DNA integrity, capacitation and recognition of pregnancy. These findings indicate that the binding of specific proteins to the plasma membrane during ejaculation may influence sperm survival after cryopreservation and may play a role in decreasing the quality in stallions with toxic seminal plasma. Elucidation of these interactions is an important step in understanding the biological processes related to equine fertility and facilitates future investigations on the selection and application of low freezability semen strategies.

Does semen quality change after local treatment of seminal vesiculitis in stallions?

Inflammation of the seminal vesicle interferes with fertility and is a persistent problem that is difficult to treat. The aim of this study was to evaluate the semen quality of 5 stallions with seminal vesiculitis before and after local treatment. All stallions were endoscopically treated for seminal vesiculitis during 10 consecutive days. The glandular lumen was accessed and flushed with a Ringer Lactate solution prior to antibiotic infusion. The antibiotic was selected based on the antibiogram from bacterial culture of samples previously collected from the seminal vesicles. The kinetic parameters (total motility - TM; progressive motility - PM; and rapid sperm - RAP), plasma membrane integrity (PMI), percentage of leukocyte (LEUK) and colony forming units (CFU) of fresh semen samples were evaluated. Additionally, nitric oxide (NO) content in seminal plasma was measured. All parameters were assessed before (T0), one week after treatment (T1) and one month after therapy (T2). The sperm kinetics and plasma membrane integrity showed an improvement in T1 that didn't last until T2. Percentage of leukocytes and CFU decreased on fresh semen and NO decreased on seminal plasma at T1 but were similar between T0 and T2. The results demonstrate that one week (T1) of local treatment leads to an improvement in sperm quality. However, this was not maintained one month (T2) after therapy, as seminal parameters at this time are similar to the pre-treatment values (T0), indicating the recurrence of the disease one month after therapy.

Paradoxical Effect of Quercetin Antioxidant on Goat Sperm Parameters After Cryopreservation.

BACKGROUND: Some antioxidants have been used in semen extenders to reduce adverse effects caused by excessive reactive oxygen species (ROS) production. The study was carried out to assess the effect of quercetin (QC) antioxidant therapy on goat semen submitted to cryopreservation. OBJECTIVE: To evaluate the effect of quercetin incorporation in different phases of the cryopreservation process of goat spermatozoa. MATERIALS AND METHODS: Five ejaculates from each of four goats (n= 20) were collected and split into four groups: Control (G1), without QC; G2, 15 µM of QC added to semen before centrifugation; G3, 15 µM QC added to semen after centrifugation; G4, 15 µM QC added to semen before centrifugation and 15 µM of QC added to semen after centrifugation (total of 30 µM of QC); and cryopreserved. All semen samples were evaluated after thawing for sperm kinetics, plasma membrane integrity, and ROS levels. RESULTS: Although lower concentrations of ROS were associated with groups that received antioxidant supplementation (P=0.0213), linear and dose dependent (P<0.05) reductions of the total and progressive sperm motility, velocity and percentage of fast cells were related to the QC groups. Likewise, plasma membrane integrity was better preserved (P=0.0154) in the control group (35.5%) than in groups that received QC (G2=32.6%, G3=32.4% and G4=26.7%). CONCLUSION: Although quercetin was efficient at reducing the oxidative stress related to sperm cryopreservation, it exerted a deleterious dose-dependent effect on the kinetics and integrity of the frozen goat semen, contradicating its use in the tested concentrations.

Protocols using detomidine and oxytocin induce ex copula ejaculation in stallions.

Tricyclic antidepressives, such as imipramine, indirectly induce ejaculation by increasing the noradrenaline concentration, which triggers an α-adrenergic response, whereas α-adrenergic agonists, such as xylazine and detomidine, directly trigger ejaculation by activating the α-1 adrenergic receptors. Furthermore, serum oxytocin concentrations in stallions increase drastically before ejaculation, but decline immediately thereafter, implicating the role of this hormone in emission. The objectives of the present study were to: 1) compare the efficiency of various protocols for inducing ex copula ejaculation in stallions, 2) evaluate the benefits of including oxytocin in the protocols, and 3) compare the semen characteristics of ex copula versus in copula ejaculates. Nine protocols were used to induce ex copula ejaculation using various combinations of xylazine (X; 0.66 mg/kg, iv); oxytocin (O; 20 IU, iv), imipramine (I; 3 mg/kg, orally), and detomidine (D; 0.02 mg/kg, iv). Imipramine was given 2 h prior to the administration of α-adrenergic agonist (detomidine or xylazine) and oxytocin. If ejaculation did not occur within 10 min after treatment with an α-adrenergic agonist, a half-dose of the same product was injected. Twelve sexually mature stallions (6-26 y) were used; 9 of 12 stallions responded to the treatment. Two stallions responded to X or XO, four stallions responded to IX and IXO, one stallion responded to DO, and five responded to IDO. Stallions that responded to detomidine did not respond to xylazine. No stallion ejaculated in response to D, ID, or IO. Erections and masturbation occurred only in imipramine-treated stallions. Sperm quality was similar among all the protocols and was not significantly different from those in in copula ejaculates collected with an artificial vagina. In a separate trial, none of these protocols induced ex copula ejaculation in 2-3 y old stallions. The side effects included sialorrhea after imipramine administration in all the stallions and sedation after administration of xylazine or detomidine. In conclusion, the new protocol, IDO, and the traditional protocol, IX, had similar results, with IDO being a useful alternative protocol in stallions for which IX was not effective. Therefore, attempts using both the protocols are encouraged, as stallions that ejaculated upon administration of detomidine did not ejaculate when xylazine was administered, whereas those that responded to xylazine did not respond to detomidine.

Influência de diferentes sistemas e curvas de congelamento na congelabilidade e fertilidade do sêmen equino / Influence of systems and freezing curve in equine semen freezability and fertility

Avaliou-se o efeito de curvas de congelação nos parâmetros espermáticos e na fertilidade, usando sêmen de alta e baixa congelabilidade. Experimento 1 - utilizou-se sêmen de quatro garanhões resistentes à congelação: grupo 1, palhetas refrigeradas até 5°C e congeladas com curva de -8°C/min; grupos 2 e 3, palhetas refrigeradas até 5°C (0,5°C/min.) e congeladas com curvas de -20°C/min e -10°C/min, respectivamente. Experimentos 2 e 3 - utilizaram-se cinco garanhões (Mangalarga Marchador), respectivamente, de alta e baixa congelabilidade: grupo 4, a mesma metodologia descrita no grupo 1; grupos 5 e 6, palhetas refrigeradas até 5°C (0,5°C/min) e congeladas com curva de -20°C/min, entre 5°C e -60°C, e -10°C/min, entre -60°C e -100ºC (grupo 5), e -25°C/min, de 5°C até -100°C (grupo 6). O sêmen foi avaliado após descongelamento pelo método computadorizado. No experimento 1, não houve diferença nos parâmetros avaliados. No experimento 2, os parâmetros motilidade total (MT) e motilidade progressiva foram superiores aos do grupo 6 em relação ao grupo 4. No experimento 3, a MT foi superior no grupo 6 em relação ao grupo 4. As curvas de congelação mais rápidas apresentaram melhores parâmetros de cinética espermática, após a descongelação, para o sêmen de garanhões da raça Mangalarga Marchador.(AU)

The effect of freezing curves on sperm parameters and fertility, using resistant and sensitive semen to cryopreservation, was evaluated. In experiment 1, Semen from 4 stallions resistant to freezing was used: Group 1, straws were cooled to 5°C and frozen with a curve of - 8°C/min; Groups 2 and 3, straws were cooled to 5°C (0.5°C/min) and frozen with curves of - 20°C / min and - 10°C/min, respectively. In experiments 2 and 3, 5 stallions (Mangalarga Marchador) presenting respectively resistant and sensitive sperm to cryopreservation were used: Group 4, same methodology described for Group 1 was performed; Groups 5 and 6, straws were cooled to 5°C (0.5°C/min) and frozen with a curve of - 20°C/min. between 5°C and - 60°C and -10°C/min. between - 60°C and - 100°C (Group 5) and - 25°C/min. 5°C to - 100°C (Group 6). Thawed-semen was evaluated by the computerized method CASA. In Experiment 1, there was no difference in the evaluated parameters. In Experiment 2, total motility (MT) and progressive motility (PM) were higher in Group 6 compared to Group 4. In Experiment 3, TM was higher in Group 6 than Group 4. The faster freezing curves showed better parameters of sperm kinetics after thawing, for the Mangalarga Marchador stallion semen.(AU)

Efeito da adição de plasma seminal oriundo de animais de alta e baixa fertilidade na criopreservação de espermatozoides da cauda do epidídimo e do ejaculado de garanhões subférteis / Effect of seminal plasma addition from high and low fertility animals on the cryopreservation of epididymal tail and ejaculated sperm from subfertile stallions

O objetivo do presente estudo foi avaliar o efeito da adição de plasma seminal de garanhões de alta e baixa fertilidade sobre a congelabilidade e a viabilidade de espermatozoides do ejaculado (EJ) e do epidídimo (EP) de garanhões subférteis. Foram utilizados seis garanhões com histórico de subfertilidade. Após coleta, espermatozoides do ejaculado foram divididos em três alíquotas: BotuSêmen® (EJ-CT); plasma seminal de alta qualidade espermática (EJ-PS1); e plasma seminal de baixa qualidade espermática (EJ-PS2). O mesmo protocolo foi realizado com espermatozoides da cauda do epidídimo após orquiectomia (EP-CT; EP-PS1; EP-PS2). Foram realizadas avaliações da cinética espermática pelo CASA e análises de integridade de membrana, acrossoma, fragmentação de DNA, capacitação espermática e peroxidação espermática por citometria de fluxo. Não foram observadas diferenças na cinética espermática entre EJ e EP, logo após a descongelação. Porém, foi observada maior (P<0,05) porcentagem de células com membranas plasmática e acrossomal íntegras nos grupos EP (EP-CT:31,7±7,5b; EP-PS1:35,2±7,0b; EP-PS2:33,9±7,2b) em comparação aos grupos EJ (EJ-CT:15,1±4,9a; EJ-PS1:11,7±4,5a; EJ-PS2:13,1±5,2a). Adicionalmente, foram observadas diferenças no índice de fragmentação de DNA (EJ-CT:2,6±0,6a; EJ-PS1:2,4±0,8a; EJ-PS2:3,0±0,8a; EP-CT:1,4±0,4b; EP-PS1:1,2±0,3b; EP-PS2:1,3±0,2b). Concluiu-se que a adição de 20% de plasma seminal, oriundo de animais férteis ou subférteis, previamente à congelação de espermatozoides epidídimários de animais subférteis não interfere na qualidade espermática.(AU)

The aim of this study was to compare the effect of the addition of seminal plasma from high and low fertility stallions on sperm viability of frozen-thawed sperm cells from ejaculate and from epididymal tail of subfertile stallions. Six stallions with a history of subfertility were used. After collection, ejaculate spermatozoa were divided into three aliquots: Botu-Semen® (EJ-CT); High-quality seminal plasma (EJ-PS1); Low-quality seminal plasma (EJ-PS2). The same was done with sperm cells from epididymis tail after orchiectomy (EP-CT; EP-PS1; EP-PS2). Evaluations of sperm kinetics were assessed by CASA and membrane and acrosome integrity, DNA fragmentation, sperm capacitation and sperm peroxidation were assessed by flow cytometry. After thawing, no differences were observed between ejaculated sperm (EJ) and epididymal sperm (EP) in any CASA evaluations. However, higher (P< 0.05) percentage of cells with intact plasma and acrossomal membranes was observed in EP groups (EP-CT:31.7±7.5b; EP-PS1:35.2±7.0b; EP-PS2:33.9±7.2b) compared to EJ groups (EJ-CT:15.1±4.9a, EJ-PS1:11.7±4.5a, EJ-PS2:13.1±5,2a). In addition, differences in DNA fragmentation index were observed (EJ-CT:2.6±0.6a; EJ-PS1:2.4±0.8a; EJ-PS2:3.0±0.8a; CT:1.4±0.4b; EP-PS1:1.2±0.3b; EP-PS2:1.3±0.2b). It was concluded that the addition of 20% seminal plasma from fertile or subfertile animals prior to the freezing of epididymal spermatozoa from subfertile animals does not interfere in sperm quality.(AU)

The effect of flunixin meglumine, firocoxib and meloxicam on the uterine mobility of equine embryos.

Embryo mobility occurs as a result of prostaglandin production by the embryo and endometrium, promoting uterine smooth muscle contractions, which propels the embryonic vesicle through the lumen. Non-steroidal anti-inflammatory drugs (NSAIDs), as flunixin meglumine, are routinely used in equine medicine and can alter the conceptus mobility if applied in early pregnancy, which may impair maternal recognition of pregnancy. The objective of this study was to evaluate and compare the effect of flunixin meglumine (FM; 1.1 mg/kg IV), firocoxib (FIRO; 0.2 mg/kg PO), and meloxicam (ML; 0.6 mg/kg, IV), on the embryo mobility. Thirty mares were divided into three groups (n = 10 per treatment). After the pregnancy diagnosis on day 12 after ovulation, the embryo mobility was evaluated by transrectal ultrasonography every 5 min for 1 h in order to visualize the location of the embryo. In all mares, three evaluations were performed: immediately before treatment (pre-treatment), after NSAID administration and 24 h after treatment. In group FM, embryo mobility decreased, from 5.8 ±â€¯0.3 movements/hour (m/h) to 2.3 ±â€¯0.5 m/h (p < 0.05) and, after 24 h the values were similar to the pre-treatment evaluation (5.9 ±â€¯0.2 m/h). Likewise, ML treatment caused a decrease of embryo movements, from 5.9 ±â€¯0.3 to 1.9 ±â€¯0.3 m/h (p < 0.05), 24 h after treatment values were 5.7 ±â€¯0.4 m/h. Treatment with FIRO did not interfere with embryo mobility (5.7 ±â€¯0.4; 5.8 ±â€¯0.3 and 5.6 ±â€¯0.3 embryo movements in the first, second and third evaluation, respectively). In conclusion, FIRO was the only NSAID that did not alter the embryo mobility and may be the safest NSAID for use in early pregnant mares.

Comparison of three different extenders on Murrah buffaloes (Bubalus bubalis) semen freezability.

The use of frozen semen for artificial insemination is the main approach utilised for the genetic improvement of most domesticated species. The advantages include lower transportation costs, continuous availability of semen, fewer occurrences of sexually transmitted diseases and the incorporation of desirable genes in a relatively short amount of time. Nevertheless, the use of frozen semen in buffalo herds remains limited due to the loss of sperm quality when buffalo semen is frozen. So, the goal of this study was to evaluate the pre- and post-cryopreservation quality of buffalo semen diluted in three distinct freezing media: Tris-egg yolk, Botu-bov® (BB) and ACP-111®. Thirty-two ejaculates from four bulls were analysed in terms of kinetics, morphology and sperm viability by epifluorescence microscope. Thawed samples were also evaluated for capacitation-like damage, DNA fragmentation and plasma and acrosomal membrane integrity using flow cytometry. The Tris-egg yolk and BB® extenders yielded better results than the ACP-111® extender for kinetics parameter (total motility, progressive motility and percentage of rapid cells). However, semen samples were similar for parameters evaluated by flow cytometry. Taken together, the data indicate that in comparison with Tris-egg yolk and BB extender, ACP-111® can also be used as an extender for buffalo semen cryopreservation.

Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation.

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer-assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.

Pentoxifylline effects on capacitation and fertility of stallion epididymal sperm.

The aims of this study were to determinate whether pentoxifylline (PTX) increases the motion parameters of fresh and frozen-thawed equine epididymal spermatozoa, to evaluate the tyrosine phosphorylation of frozen-thawed epididymal sperm in the presence of PTX and to determine whether the PTX-treatment of stallion epididymal sperm prior to freezing improves the fertility response of mares to a reduced number of spermatozoa per insemination dose. Fifty epididymis were flushed with a skim milk based extender with or without PTX. The pre-treatment with PTX enhanced the sperm motility after being harvested (P<0.05); however the freeze-thaw process did not alter the sperm kinematics between control and treated samples (P>0.05). Plasma membrane integrity did not differ between control and PTX group after recovery and after thawing (P>0.05), as observed in tyrosine phosphorylation, which the PTX treatment did not alter the percentage of tail-associated immunofluorescence of cryopreserved epididymal sperm (P>0.05). For the fertility trial, different insemination groups were tested: 800×106 epididymal sperm (C800); 100×106 epididymal sperm (C100); 100×106 epididymal sperm recovered in an extender containing PTX (PTX100). The conception rates for C800; C100 and PTX100 were 68.7% (11/16); 31.5% (5/16) and 50% (8/16), respectively. The conception rate did not differ among groups (P>0.05), however, a low number of animals was used in this study. A trend toward significance (P=0.07) was observed between C800 and C100 groups. In conclusion, PTX has no deleterious effect on sperm motility, viability and capacitation of cryopreserved stallion epididymal sperm. The conventional artificial insemination with 100×106 sperm recovered with PTX ensures acceptable conception rates and maximize the limited number of doses of cryopreserved stallion epididymal sperm.

Seminal plasma arising from the whole boar sperm-rich fraction increases the stability of sperm membrane after thawing.

Boar spermatozoa arising from the sperm-rich ejaculate fraction are reported to have a more stable plasma membrane and are more resistant to cold shock and premature acrosome reaction than spermatozoa from the whole ejaculate. Furthermore, seminal plasma (SP) can increase the cryotolerance of boar spermatozoa, and in other domestic species, it has the ability to reverse cryopreservation damage. This study aimed to evaluate the effects of boar SP arising from the whole sperm-rich ejaculate fraction (SP-SRF) on the integrity, stability, and peroxidation of sperm membranes after thawing. Each ejaculate ( = 24) was divided among 4 treatments: control (CT), centrifuged and suspended in autologous SP-SRF (CS), centrifuged with withdrawn SP-SRF (CW), and post-thawed SP arising from the whole sperm-rich fraction addition to CW (CWSP). After thawing, all treatments were incubated for 5, 60, and 120 min and were analyzed for membrane integrity, fluidity, and peroxidation by flow cytometer. The absence of SP-SRF increased the lipid disorder ( < 0.05) but had no effect on lipid peroxidation ( > 0.05) or membrane integrity ( > 0.05). However, the increase in lipid disorder by withdrawal of SP-SRF was reversed by SP-SRF addition ( < 0.05) to the post-thawing medium, whereas plasma and acrosomal membrane integrity ( > 0.05) and lipid peroxidation ( > 0.05) were unchanged. In conclusion, despite the centrifugation effects, the addition of SP arising from the whole sperm-rich fraction to post-thawed boar semen decreased sperm lipid disorder without an influence of the sperm membrane integrity and peroxidation.

Sperm fertility and viability following 48h of refrigeration: evaluation of different extenders for the preservation of bull semen in liquid state.

Two experiments were conducted to compare the effectiveness of different extenders conventionally used for semen cryopreservation to maintain the viability and fertility of cooled bull semen. In Experiment 1, sperm samples obtained from 20 Nellore bulls were preserved at 5°C for 48h using two extenders containing 20% of egg yolk [Tris (TRIS-R) and Botu-Bov(®) (BB)] and another composed of 1% soy lecithin [Botu-Bov(®)-Lecithin (BB-L)] as substitutes for animal origin products. The samples were evaluated at 6, 24 and 48h for plasma and acrosomal membrane integrity, quantification of thiobarbituric acid reactive substances (ng of TBARS/10(8) cells) and sperm motility parameters by computer-assisted semen analysis (CASA). In Experiment 2, pregnancy rate (P/AI) of 973 fixed-time artificially inseminated Nellore cows were compared when cows were inseminated with conventionally cryopreserved semen in TRIS-egg yolk glycerol (TRIS-C Control, n=253) or semen cooled for 48h in TRIS-R (n=233), BB (n=247) or BB-L (n=240). Although none of the extenders used was effective on maintaining total progressive motility and cellular integrity throughout the 48-h of the refrigeration period (P<0.01), BB-L conferred greater protection against oxidative stress (P<0.05) than egg yolk-based medias. The P/AI for semen samples preserved in TRIS-C, TRIS-R, BB and BB-L were 39.92(a), 25.32(b), 26.32(b) and 33.33(ab), respectively. These results demonstrate that the three conventional extenders used for semen cryopreservation do not provide the protection required to maintain bull semen fertility under refrigeration for a 48-h period, resulting in reduced pregnancy rates. However, the use of lecithin-based medium instead of egg yolk results in greater protection against lipid peroxidation, producing P/AI results comparable to those obtained using frozen semen.

Use of cholesterol-loaded cyclodextrin: an alternative for bad cooler stallions.

During the cooling process, sperm may suffer irreversible damage that compromises the fertility rate. Incorporating cholesterol-loaded cyclodextrin (CLC) represents a strategy to increase sperm resistance at low temperatures; however, high levels of cholesterol in the cell membrane can interfere with sperm capacitation. The goals of this study were to determine the CLC concentration and cooling temperature that produce optimal kinetic parameters and viability of sperm from stallions identified as bad coolers (BCs) and good coolers (GCs), as well as the effect of adding CLC on the occurrence of the acrosome reaction (ACR) and on the fertility rate of cooled sperm. In experiment 1, each ejaculate was divided into four groups: Control and treated with 1 (CLC-1), 1.5 (CLC-1.5), or 2 mg (CLC-2) of CLC/120 × 10(6) sperm and cooled for 48 hours at 5 °C. In experiment 2, each ejaculate was divided into four groups: Control and CLC-1.5 cooled at 15 °C or 5 °C for 24 hours. For experiment 3, GC and BC stallions were used, and the ejaculates were divided into control and CLC-1.5 cooled at 5 °C for 48 hours. According to experiment, the sperm kinetics (SK) and plasma membrane integrity (PMI) were analyzed before and after 24 and 48 hours of cooling. In experiment 4, the ejaculates were divided into four groups: Control and CLC-1.5 maintained at room temperature or cooled at 5 °C for 24 hours. Each group was incubated with ionophore calcium at 37 °C for 3 hours. The incidence of ACR was analyzed before and after 1, 2, and 3 hours of incubation. For experiment 5, two cycles of 10 mares for a GC stallion and two cycles of 25 for a BC stallion were used. The inseminations were performed with control and CLC-1.5 groups cooled at 5 °C for 24 hours. According to results, all groups treated with CLC exhibited higher PMI compared with controls, and CLC-1.5 and CLC-2 exhibited the best SK results. The cooling temperature of 5 °C was superior to 15 °C when the sperm was treated with CLC. The GC and BC stallions benefited from the CLC-1.5 treatment, but the BCs were more evident, which presented greatly increased PMI and SK. There was a delay in capacitation of at least 3 hours for the fresh sperm and at least 1 hour for cooled sperm supplemented with CLC-1.5. After adding CLC-1.5, the fertility of BC stallion significantly increased, but that of the GC was not altered. Thus, incorporating CLC is an effective technique to cool equine semen, although it is indicated mainly for BC stallions.

Cooling of ejaculated and epididymal stallion sperm / Refrigeração de espermatozoides do ejaculado e epidídimo em garanhões

After a serious injury or sudden death, epididymis cauda sperm recovery and cryopreservation may present as the last opportunity to obtain genetic material from a valuable stallion. This study evaluated the viability of cooled equine sperm collected by three different methods: sperm of ejaculated (G1), sperm recovered from the epididymal cauda immediately after orchiectomy (G2) and sperm recovered from the epididymal cauda after storage for 24 hours at 5°C (G3). To obtain G1 sperm, two ejaculates were collected. After 1 week, all stallions underwent a bilateral orchiectomy, and one of the removed epididymides was flushed to obtain G2 sperm. The contralateral epididymis was stored at 5°C for 24 hours before being flushed to obtain G3 sperm. The sperm samples were evaluated immediately after the addition of the refrigeration extender, and after 24 and 48 hours of storage at 5°C. After 24 and 48 hours of storage, the epididymal sperm demonstrated higher motility traits when compared to the ejaculated sperm (P<0.05). These results indicate that sperm recovered from the epididymal cauda of stallions are more resistant to the cooling process, with higher kinetic parameters and plasma membrane integrity when compared to the ejaculated sperm.

A recuperação de espermatozoides da cauda do epidídimo pode ser a última chance para preservação do germoplasma quando ocorre morte súbita ou lesão grave em garanhões de alto valor genético. O presente trabalho comparou a viabilidade após refrigeração dos espermatozoides do ejaculado (G1), recuperados da cauda do epidídimo imediatamente após a orquiectomia (G2) e recuperados após armazenamento do epidídimo por 24 horas a 5ºC (G3). No G1 foram colhidos dois ejaculados. Uma semana após a colheita dos ejaculados os garanhões foram submetidos à orquiectomia bilateral e realizada a colheita dos espermatozoides da cauda do epidídimo de um testículo de cada garanhão (G2). O testículo contralateral permaneceu a 5°C por 24 horas, antes da recuperação espermática (G3). A análise das amostras foi realizada imediatamente após a adição do meio de refrigeração, e após 24 e 48 horas de armazenamento a 5°C. Após 24 e 48 horas de armazenamento, os espermatozoides do epidídimo demonstraram características de cinética maiores que os do ejaculado (P<0.05). Estes resultados indicam que espermatozoides recuperados da cauda do epidídimo foram mais resistentes ao processo de refrigeração, com maiores parâmetros de cinética espermática e integridade da membrana plasmática quando comparados aos espermatozoides do ejaculado.

New seminal plasma removal method for freezing stallion semen.

Seminal plasma removal, an indispensable step in equine semen cryopreservation, is usually done by centrifugation, but this might cause mechanical damage to sperm. A new method for seminal plasma removal from stallion semen, namely a filter composed of a synthetic hydrophilic membrane (Sperm Filter, BotuPharma, Botucatu, Sao Paulo, Brazil), was recently proposed. The objective of this study was to test the use of the Sperm Filter in the removal of seminal plasma before freezing stallion semen. Ejaculates from 31 stallions were divided into two groups and cryopreserved. In group 1 (G1), seminal plasma was removed with the Sperm Filter, and in group 2 (G2), seminal plasma was removed by centrifugation (600×g for 10 minutes). There were no differences (P < 0.05) between G1 and G2 in sperm kinetic parameters or plasma membrane integrity before or after cryopreservation. However, sperm recovery rate was higher (P < 0.05) for G1 versus G2 (mean ± SD, 89.4 ± 7.4% vs. 80.9 ± 5.5%). Therefore, the Sperm Filter was as efficient as centrifugation in removing seminal plasma from the stallion ejaculate. However, filtering was more practical and had significantly fewer sperm lost than the centrifugation technique.

Induction of double ovulation in mares using deslorelin acetate.

This study aimed to determine whether deslorelin acetate could induce double ovulation in mares. In Experiment 1, eight mares were treated with prostaglandin on Day 8 (D8) after ovulation, then treated with saline or with 100 µg of a controlled-release formulation of deslorelin acetate vehicle intramuscularly (IM) every 12h from D8 after ovulation until at least two follicles reached 33 mm. At this time, ovulation was induced with 2500 IU of hCG. Artificial insemination was performed 24h after induction, and embryos were collected on the eighth day after ovulation was first detected. In Experiment 2, 112 estrous cycles in 56 mares were studied. In this experiment, the deslorelin acetate protocol was initiated only in mares that achieved a follicle with a diameter of at least 25 mm and at least one second follicle with a diameter≥20mm was detected, at which time 100 µg deslorelin acetate or saline was administered IM every 12h. The other procedures were similar to those described in Experiment 1. The variables studied were analyzed using Student's t-test and Fisher's exact test. In Experiment 1, only two mares in deslorelin group having second follicles of 20-25 mm on responded with double ovulation. In the second experiment, 82% of treated mares responded with double ovulation, and the embryo recovery per estrous cycle was 1.12 and 0.57 in the group treated with deslorelin acetate and the control group, respectively (P<0.05). Deslorelin acetate is effective in inducing double ovulation in mares using the protocol proposed. On average, it allows for the recovery of one embryo by uterine flushing.

Cryopreservation and fertility of ejaculated and epididymal stallion sperm.

The cryopreservation of epididymal sperm is important to preserve genetic material from valuable deceased males. This study evaluated the viability of sperm samples from eight stallions under three conditions: (1) collected using an artificial vagina (EJ-0h), (2) recovered from the epididymal cauda immediately after orchiectomy (EP-0h), and (3) recovered from the epididymal cauda after 24h of storage at 5°C (EP-24h). To obtain EJ-0h sperm, two ejaculates were collected from each stallion. After 1 week, the stallions were submitted to bilateral orchiectomy, and one of the removed epididymides was flushed to obtain EP-0h sperm. The contralateral epididymis was stored at 5°C for 24h before being flushed to obtain EP-24h sperm. The sperm samples were analyzed at three different times: immediately after sperm recovery, after dilution in the freezing extender, and post-thawing. A fertility trial was performed using 39 estrous cycles. After ovulation induction with 1mg of deslorelin acetate (i.m.), mares were inseminated with 800×10(6) sperm. The total number of sperm recovered was 7.8±4.7×10(9) for EJ-0h sperm, 12.9±9.2×10(9) for EP-0h sperm and 12.0±8.0×10(9) for EP-24h sperm. The sperm motility, evaluated by total motility, progressive motility and the percentage of rapid cells, was similar among the samples before and after freezing (P>0.05). However, the plasma membrane integrity was different between EJ-0h and EP-0h pre-freezing and between EJ-0h and EP-24h post-thawing (P<0.05). The conception rates were similar between groups inseminated with sperm recovered from the epididymal cauda immediately after orchiectomy (EP-0h), after 24h of storage at 5°C of the epididymal cauda (EP-24h) and with ejaculated sperm (EJ-0h) (P>0.05). In conclusion, the viability and fertility of cauda epididymal sperm are similar to those of ejaculated sperm.

Effect of progesterone and ionomycin on domestic cat sperm motility patterns and acrosome reaction.

This study was aimed at assessing the changes in sperm motion patterns and the percentage of acrosome reaction (AR) in domestic cat semen after treatment with either ionomycin or progesterone (P(4)). Ten ejaculates were collected from five tomcats using an artificial vagina, and were diluted, centrifuged and resuspended in a capacitation medium. Samples were evaluated and divided into seven equal aliquots and, after 2 h at 25 degrees C, were incubated for 30 min at 38 degrees C in 5% CO(2) and then analyzed. Computer-assisted sperm analysis and a combination of three fluorescent probes were used to assess sperm plasma, acrosomal membrane integrity and mitochondrial transmembrane potential. Thirty minutes after the start of incubation, P(4) was added (10 microg/ml) to the P1 group. Groups P2 and P3 were supplemented with P(4) (10 and 20 microg/ml, respectively) only after 2 h of incubation, and groups I1 and I2 were supplemented with ionomycin (4 and 8 mum, respectively) 2 h after incubation. Group E was supplemented with ethanol (0.6%) at 2 h after incubation and group C received no supplementation. Ionomycin and P(4) treatments led to a hyperactivation-like sperm motion and an increase (p < 0.05) in the percentage of AR. Although a higher (p < 0.05) percentage of AR was obtained in group I2 when compared with all P(4) groups, a decrease (p < 0.05) in total and progressive motility was observed in I2 group. As I1 group was similar to I2 to induce AR without diminishing sperm motility, we can conclude that ionomycin at 4 microm seems to be more suitable to trigger AR in domestic cat sperm.

Thermoresistance sperm tests are not predictive of potential fertility for cryopreserved bull semen.

Different studies demonstrate positive correlations between seminal variables determined in the laboratory and subsequent fertility after artificial insemination. It is clear, however, that there is still a deficiency in predicting in vivo fertility results of semen samples. The present study intended to verify the efficiency of rapid and slow thermoresistance tests in predicting fertility of frozen semen of bulls. Sperm from 64 ejaculates of 39 Nelore bulls (Bos indicus), aged 2-10 years, were cryopreserved in 0.5 mL straws. Thawed straws containing 30 x 10(6) sperm were analyzed for seminal variables in the laboratory and used to inseminate 4920 cows to evaluate fertility in the field. The ejaculates were frozen in a Tris-based extender and samples were evaluated for total motility after rapid (46 degrees C/30 min) and slow (38 degrees C/5h) thermoresistance tests by conventional and computerized (CASA) methods. Sperm samples were grouped according to their ability to retain motility after thermoresistance testing: group 0 (0% motility), group 1 (1-20% total motility), group 2 (21-40% total motility) and group 3 (>40% total motility). Correlation and association between these groups and fertility diagnosed by rectal palpation at 90 days were verified. Chi-square test demonstrated no association between motility groups and fertility (P>0.25) and both rapid and slow thermoresistance tests had a lesser correlation to fertility (r=0.11 and 0.14, respectively). These results demonstrated that these tests are not reliable in predicting in vivo behavior of bull frozen semen and are not effective to estimate fertility.

Freezing of stallion epididymal sperm.

Inseminations with frozen-thawed epididymal sperm have resulted in low-pregnancy rates of mares. If fertility of epididymal sperm could be improved, it would help to preserve genetic material from stallions that have suffered severe injuries, been castrated or have died. The aim of the present study was to investigate the effect of different extenders and pre-freezing addition of capacitation media on freezability of epididymal sperm and on storage at 5 degrees C for 24h. In experiment 1, epididymal sperm samples were diluted and subsequently frozen with three different extenders: Botu-Crio, EDTA-Lactose and INRA-82. Motility analysis using computer assisted sperm analyzer (CASA) demonstrated better motility for sperm in Botu-Crio than in the other extenders; EDTA-Lactose yielded better motility than INRA-82 on most evaluated parameters. There was no difference in membrane integrity among the studied extenders. From 18 inseminated mares, 12 (66%) were pregnant 15 days after AI with frozen-thawed epididymal sperm showing that Botu-Crio was able to maintain the fertility potential. In experiment 2, the effect of incubation of epididymal sperm before freezing in three capacitation media (Fert Talp, Sperm Talp, Talp+Progesterone), seminal plasma, or control was tested. Based on post-thaw motility evaluation by CASA, samples incubated in Sperm Talp showed better motility values. There were no differences in plasma or acrosomal membranes or in mitochondrial potential among groups. We concluded that Botu-Crio was better than the other extenders in the ability to preserve epididymal sperm and that pre-freeze addition of Sperm Talp was also beneficial.