Seminal plasma removal for medium-term preservation of ram sperm at 5 °C.

This study aimed to investigate if washing ram sperm from seminal plasma (SP) could be an effective tool to extend sperm lifespan in medium-term preservation in liquid form to optimize ovine artificial insemination protocols. To this end, in Experiment 1 SP was added to a sperm model without previous contact with this substance (ram epididymal sperm) at the beginning or the end of a 48-hour preservation protocol at 5 °C (n = 13). Sperm motility and kinetic parameters and sperm functionality in terms of sperm viability, apoptosis, mitochondrial activity and reacted acrosomes were assessed after 6 h of storage at 15 °C (standard liquid preservation method) and 24 and 48 h at 5 °C. Extended sperm showed better results after 48 h when stored in the absence than in the presence of SP in most sperm quality parameters. Moreover, the final SP supplementation of this experimental group resulted in the highest sperm motility and kinetic parameters, viability and mitochondrial activity. These results suggested that initial SP deprivation could be beneficial in a medium-term ram sperm preservation protocol in liquid form, as well as a final supplementation. Therefore, we conducted Experiment 2 to evaluate the effect of SP removal from freshly ejaculated ram semen under the same storage conditions as in Experiment 1 (n = 12). Surprisingly, SP withdrawal impaired sperm functionality, leading to increased apoptosis and decreased mitochondrial activity after 24 and 48 h at 5 °C. Conversely, SP supplementation at the end of the preservation protocol of the ejaculate processed as usual had a positive effect on sperm quality and fertility. To summarize, SP absence was beneficial for a medium-term preservation protocol (up to 48 h at 5 °C) of ram epididymal sperm, but the same preservation protocol for ram ejaculated sperm revealed a possible failure of the SP removal method in avoiding the sperm-SP interaction effect. Meanwhile, SP supplementation of ram semen at the end of the preservation protocol increased in vitro sperm quality and fertility after artificial insemination.

Unveiling the Potential of Aloe vera Gel Supplementation in a Cooling Extender: A Breakthrough in Enhancing Rooster Sperm Quality and Fertility Ability.

The cooling of semen storage at 5 °C from a Thai native rooster (Pradu Hang Dum), supplemented with herbs possessing antioxidant properties, provided limited research. This study was conducted to evaluate the efficiency of Aloe vera (AV) gel supplementation at various levels on the quality of cooled semen and subsequent fertility after artificial insemination. Sixty-four chickens had semen pooled, diluted, and supplemented with different levels of AV gel (0% as control, 0.25%, 0.50%, 1.0%, 2.5%, 5.0%, 10%, and 20%), and then stored for 72 h. In Experiment 1, semen quality, malondialdehyde (MDA) levels, and pH values were assessed at 0, 24, 48, and 72 h after storage. Experiment 2 assessed fertility potential using the most effective cooled storage semen from Experiment 1. Results showed a decrease in semen quality with prolonged storage time (p < 0.001). The highest semen quality was observed in the group supplemented with 1.0% AV gel (p < 0.001), whereas the lowest was noted in the 20% AV gel group (p < 0.001). Furthermore, the 1.0% AV gel group exhibited the highest semen quality at 24, 48, and 72 h of storage. The evaluation of fertility and hatchability rates revealed a statistically significant improvement in fertility potential (p < 0.05) in the group supplemented with 1.0% AV gel. In summary, this study represents the first investigation of stored Thai native rooster semen using a semen extender supplemented with Aloe vera gel at 5 °C, demonstrating its efficacy for storage up to 72 h. The addition of 1% AV gel was recommended as an antioxidant supplementation during the semen storage process at 5 °C to enhance semen quality and fertility rates.

Post-cooling sperm processing can rescue sperm quality of cooled-stored stallion semen.

Poor sperm quality in cooled-shipped semen has been related to subpar fertility in horses. Therefore, this study aimed to evaluate the ability of post-cooling sperm processing to improve sperm parameters of cooled-stored stallion semen for artificial insemination. For all experiments, ejaculates were collected, processed, and diluted in skimmed milk-based (SM) medium and stored at 5 °C/24h. In all experiments an aliquot of unprocessed cooled semen was used as a control. In the first experiment (Exp 1.), cooled-stored semen from 16 stallions (n = 32) was processed by SpermFilter or centrifugation (600×g/10min) and resuspended in an egg yolk-based freezing medium containing permeating cryoprotectants (EY-C) for cryopreservation. Sperm recovery and motility parameters were immediately assessed after sperm resuspension in both groups and compared with unprocessed (Unp) samples. In Exp 2., cooled semen samples from six stallions (n = 18) were processed using SpermFilter and resuspended in SM or EY-C. Motility parameters and plasma membrane integrity were assessed in all groups (Unp, SM, and EY-C). In Exp 3, cooled semen from four stallions (n = 20) was processed by SpermFilter, resuspended in SM, EY-C, or egg yolk-based medium without cryoprotectants (EY-nC); and submitted to a thermoresistance test (37 °C/3h). Motility parameters, plasma membrane integrity and stability, mitochondrial membrane potential, mitochondrial superoxide generation, and DNA fragmentation index were evaluated in all groups. Finally, in Exp 4, 39 estrous cycles of 11 mares were inseminated with unprocessed (n = 6) cooled-stored semen or semen cooled at 5 °C/24h and then processed by SpermFilter and resuspended in SM (n = 5), EY-C (n = 11), EY-nC (n = 11), or centrifuged and resuspended in EY-C (n = 6). Overall, semen processing and resuspension in EY mediums (EY-C and EY-nC) improved sperm parameters compared with those of unprocessed semen (P < 0.05). Centrifugation (91 ± 5 %) recovered more sperm than SpermFilter (84 ± 9 %; P < 0.05). Sperm resuspended in EY-nC maintained better sperm parameters throughout the thermoresistance test than those in the other groups (P < 0.05). The fertility rates were similar between all groups (P > 0.05). In conclusion, processing and resuspension in EY medium can improve sperm parameters in post-cooled-stored stallion semen.

Ovine fertility by artificial insemination in the breeding season could be affected by intraseasonal variations in ram sperm proteomic profile.

It is important to note that seasonality could affect ram reproductive parameters, and therefore, fertility results after artificial insemination. In this work, 1) we assessed fertility rates after cervical artificial insemination of 11,805 ewes at the beginning (June 21st to July 20th) and at the end (November 20th to December 21st) of the reproductive season in the Assaf breed for the last four years, and 2) we aimed to identify male factors influencing the different reproductive success obtained depending on the time at the mating season in which ovine artificial insemination was performed. For this purpose, we evaluated certain ram reproductive and ultrasonographical parameters as well as we performed a multiparametric and proteomic sperm analysis of 6-19 rams at two very distant points in the mating season (July as Early Breeding Season -EBS- and November as Late Breeding Season -LBS-). Rutinary assessments carried out in the ovine reproduction centers (testicular volume, libido, sperm production and mass motility) showed non-significant differences (P ≥ 0.05) between both studied times, as well as the ram ultrasonographic evaluation (Resistive and Pulsatility Index as Doppler parameters; and pixels mean gray level, and hypoechoic areas percentage and density as echotexture parameters). However, at level of sperm functionality, although sperm quality appeared non-significantly lower (P ≥ 0.05) in the EBS, we identified a significantly different (P < 0.05) sperm proteomic profile between the seasonality points. The following proteins were identified with the lowest abundance in the EBS with a fold change > 4, a P = 2.40e-07, and a q = 2.23e-06: Fibrous Sheath-Interacting Protein 2, Disintegrin and Metalloproteinase Domain-Containing Protein 20-like, Phosphoinositide-Specific Phospholipase C, Tektin 5, Armadillo Repeat-Containing Protein 12 Isoform X3, Solute Carrier Family 9B1, Radial Spoke Head Protein 3 Homolog, Pro-Interleukin-16, NADH Dehydrogenase [Ubiquinone] 1 Alpha Subcomplex Subunit 8, Testis, Prostate and Placenta-Expressed Protein, and Acyl Carrier Protein Mitochondrial. In conclusion, while our basic analyses on male and sperm quality showed similar results between the beginning and the end of the breeding season, on a proteomic level we detected a lower expression of sperm proteins linked to the energy metabolism, sperm-oocyte interactions, and flagellum structure in the EBS. Probably, this different protein expression could be related to the lower fertility rate of Assaf ewes after cervical artificial insemination at this time. More importantly, sperm proteins can be used as highly effective molecular markers in predicting sperm fertilization ability related to intraseasonal variations.

An optimized centrifugation protocol for ram sperm ensuring high sample yield, quality and fertility.

The optimization and implementation of artificial insemination (AI) in sheep is necessary to increase the livestock productivity through enhanced control of reproductive function. Sperm centrifugation is a common procedure in the ejaculate handling in AI and other assisted reproductive technologies (ART), as part of new methods of sperm analysis, selection or preservation. However, our research group previously established that this simple procedure might cause a large sperm loss and induce deleterious effects on the sperm function of the ovine species when high centrifugation forces are employed. To our knowledge, there are no studies on combined effect of extender and different centrifugal forces on ram sperm yield and quality. Furthermore, evidence of in vivo fertility rate using sperm obtained with various centrifugation forces is also lacking in this species. Thus, the objective of this work was to define the ideal conditions for ram semen centrifugation that will achieve the best quantity and quality sample to ensure unaffected fertilization ability of centrifuged ram sperm. The Experiment 1 evaluated the effect of the centrifugation procedure of two extenders (INRA 96 and Tyrode's) and two cooling protocols (Rapid and Slow Refrigeration -35 °C to 15 °C-) on sperm recovery rate and quality (motility and kinetic parameters, viability, apoptosis and mitochondrial activity). INRA 96 combined with Slow Refrigeration and Tyrode's at room temperature registered the highest sperm recovery and quality values (P ≤ 0.05). In Experiment 2, the influence of three centrifugal forces (600, 1200 and 6000×g for 10 min) was assessed immediately after centrifugation on the technical performance and sperm functionality in diluted samples with INRA 96 and Tyrode's at the conditions set out in Experiment 1. The lowest pellet weight (P ≤ 0.05) without harmful effect on sperm physiological status (P > 0.05) was achieved at 1200×g, since 6000×g induced sperm motility damage (P ≤ 0.05) with both extenders. Finally, to ensure the total safety of the centrifugation protocol, Experiment 3 tested in a combined in vitro and in vivo test the effect of these three centrifugal forces on ram sperm quality after dilution (INRA 96) and liquid storage (6-8 h at 15 °C). The damage produced by 6000×g on sperm motility (P ≤ 0.05) was maintained over time, coinciding with a lower fertility (P ≤ 0.05). In conclusion, ram sperm can be centrifuged in INRA 96 extender up to 1200×g for 10 min at 15 °C as secure values with high recovery rates and without detrimental effects on sperm quality and fertility.

Effects of boar sperm antioxidant supplementation on fertility.

Use of cooled stored boar semen for artificial insemination accounts for 99% of inseminations in pork production sectors worldwide. The aim of the present study was to examine use of GameteGuard®-CP, a natural plant derived blend of antioxidants, supplemented in one commercially available semen extender on sperm quality, conception rate, farrowing rate, and litter size. Ejaculates from 16 commercial Duroc boars were used in a split-ejaculate single-sire artificial insemination study. Sperm were evaluated for motility, viability, acrosome integrity, membrane stability, mitochondrial membrane potential, DNA intactness, and total antioxidant reactivity on both day 1 and 4 subsequent to ejaculate collection. Sows were inseminated after a treatment regimen was imposed to synchronize time of ovulation (n = 1476) among females using sperm collected 4 days subsequent to ejaculate collection extended in AndroStar Plus® or GameteGuard®-CP supplemented extender. At day 4 post-ejaculate collection, there was maintenance of sperm viability, membrane stability, acrosome intactness, mitochondrial membrane potential, and DNA intactness (P < 0.01) when there was use of GameteGuard®-CP treatments. When control samples were used, there were lesser (P < 0.01) values from day 1-4 for all variables evaluated. Conception rate and farrowing rates were 7.4% and 9.7% greater, respectively (P < 0.05), but there was no difference as a result of GameteGuard®-CP-treatment in mean litter outcomes. The results in the present study indicate antioxidant supplementation using GameteGuard®-CP maintains sperm quality during cool sperm storage resulting in improved conception and farrowing rates when using semen stored in the cooled state for as long as 4 days.

Cooled storage of semen from livestock animals (Part II): Camelids, goats, and sheep.

This review is part of the Festschrift in honor of Dr. Duane Garner and provides an overview of current techniques in cooled storage of semen from livestock animals such as camelids, goats, and sheep. Facing worldwide environmental changes and a trend towards more conscious and healthy eating behaviors, the development of a stable animal breeding industry is a significant challenge for the near future. In the present review, factors influencing semen handling in camelids, goats and sheep are described and relevant methods as well as current trends to improve liquid-storage of cooled semen are discussed, including extenders, additives, cooling rates, and storage temperatures. The species-specific physiology and resulting challenges are taken into consideration. While the main problem for camelid semen processing is the relatively greater viscosity as compared with that of some other animals, the deciding factor for successful artificial insemination (AI) in goats and sheep is the site (i.e., cervical or vaginal) of semen placement in the reproductive tract. Due to the type of cervical anatomy, the penetration of the cervix when using AI instruments is rather difficult. Furthermore, the seminal plasma of small ruminants affects the interaction with milk-based extenders and egg yolk which results in species-specific regimens for cooled liquid-preservation. Comparing all three species, the greatest pregnancy rates were obtained by AI with goat semen after cooled liquid-storage for several days.

Use of cooled buffalo semen as a strategy to increase conception rates in fixed-time artificial insemination programs during unfavorable reproductive periods / [Uso de sêmen resfriado de búfalo como estratégia para aumentar as taxas de concepção em programas de inseminação artificial em tempo fixo, durante períodos reprodutivos desfavoráveis]

The objective of this study was to compare the reproductive efficiency of dairy buffaloes undergoing fixed-time artificial insemination (FTAI) protocols based on progesterone/estrogen (P4/E2) and eCG during unfavorable breeding season using cooled (CS) and frozen semen (FS). A total of 446 buffaloes (> 40 days postpartum) were randomly distributed into four blocks (years): B1-2014 (n = 143), B2-2015 (n = 34), B3-2016 (n = 90), and B4-2017 (n = 179). Each block was subdivided into two (AI with CS and FS using the same ejaculate of each bull). Thus, the block subdivision was as follows: B1 (CS = 71 and FS = 72); B2 (CS = 18 and FS = 16); B3 (CS = 47 and FS = 43); and B4 (CS = 90 and FS = 89). The ejaculates of eight Murrah bulls collected using an artificial vagina were divided into two aliquots: one aliquot was diluted in Botu-Bov® commercial extender and cooled (BB-CS), and the other was diluted in the same extender and frozen (BB-FS). BB-CS aliquots were cooled at 5 °C/24 h using a refrigerator. BB-FS group aliquots were also cooled, and after equilibrating at 5 °C for 4 h, were placed in a 21-L Styrofoam box, 5 cm above the surface of liquid nitrogen. In the afternoon (A) on D0 (2:00 p.m.) the animals received EB 2.0 mg IM (Estrogin®) and an ear implant (CRESTAR® 3.0 mg P4). At D9 (A), the implant was removed, and the animals received eCG 400 IU IM (Folligon® 5000) + Cloprostenol PGF2α 0.530 mg IM (Sincrocio®). At D10 (A), the animals received EB 1.0 mg IM (Estrogin®), and at D12 (8:00 a.m.), AI was performed. At D42, pregnancy was diagnosed via ultrasonography. Total CRs were 48.2% CS and 34.6% FS for years 2014 to 2017, with a significant difference of 13.7% (P<0.05). In conclusion, cooled semen resulted in higher CR than frozen semen in dairy buffaloes under the P4/E2 and eCG FTAI during the unfavorable reproductive season.(AU)

O objetivo deste estudo foi comparar a eficiência reprodutiva de búfalas leiteiras submetidas a protocolos de inseminação artificial em tempo fixo (IATF) à base de progesterona/estrogênio (P4/E2) e eCG, durante a estação reprodutiva desfavorável, usando-se sêmen resfriado (SR) e congelado (SC) Um total de 446 búfalas (> 40 dias após o parto) foi distribuído aleatoriamente em quatro blocos (anos): B1-2014 (n = 143), B2-2015 (n = 34), B3-2016 (n = 90) e B4-2017 (n = 179). Cada bloco foi subdividido em dois (IA com SR e SC utilizando-se a mesma ejaculação de cada touro). Assim, a subdivisão do bloco foi a seguinte: B1 (SR = 71 e SC = 72); B2 (SR = 18 e SC = 16); B3 (SR = 47 e SC = 43); e B4 (SR = 90 e SC = 89). Os ejaculados de oito touros Murrah coletados com vagina artificial foram divididos em duas alíquotas: uma alíquota diluída em diluente comercial Botu-Bov® e resfriada (BB-SR), e a outra diluída no mesmo diluente e congelada (BB-SC). As alíquotas de BB-SR foram resfriados a 5°C/24h usando-se um refrigerador. As alíquotas do grupo BB-SC também foram resfriadas e, após equilíbrio a 5°C por 4h, foram colocadas em uma caixa de isopor de 21L, 5 cm acima da superfície do nitrogênio líquido. À tarde (A), no D0 (14h), os animais receberam BE 2,0 mg IM (Estrogin®) e um implante auricular (Crestar® 3,0 mg P4). No D9 (A), o implante foi retirado e os animais receberam eCG 400 UI IM (Folligon® 5000) + cloprostenol PGF2α 0,530 mg IM (Sincrocio®). No D10 (A), os animais receberam BE 1,0mg IM (Estrogin®), e, no D12 (8h da manhã), foram realizadas as IAs. No D42, a gestação foi diagnosticada por ultrassonografia. As taxas de concepção (TC) totais foram 48,2% SR e 34,6% SC para os anos de 2014 a 2017, com uma diferença significativa de 13,7% (P<0,05). Em conclusão, o sêmen resfriado resultou em maior TC do que o sêmen congelado em bubalinos leiteiros sob P4/E2 e eCG FTAI durante a estação reprodutiva desfavorável.(AU)

Use of nutraceuticals in the stallion: Effects on semen quality and preservation.

Nutritional supplements are widely used in the equine industry with the aim of improving horse health, sports or reproductive performances. Over the years, a number of studies have focused on investigating the effects of several dietary compounds on the quality and preservation of stallion semen. This paper reviews the literature available on the use of nutritional supplementation for the improvement of reproductive performance and semen quality in equine species, critically appraising the benefits and negative effects of several compounds found in complementary feeds such as PUFAs from different sources, vitamins and antioxidants, carnitine and botanical extracts. Different nutraceuticals have been highlighted to improve stallion fertility by providing optimal levels of antioxidants, with the most promising results obtained by the combination of PUFAs and antioxidants that resulted to be essential for the maintenance of normal reproductive functions and the reduction of cryodamage in cooled and frozen equine semen.

Effect of egg yolk-based extender and seminal plasma removal on sperm viability of cooled donkey semen / Efeito do diluente à base de gema de ovo e remoção do plasma seminal na viabilidade espermática do sêmen refrigerado de jumento

Developing effective cooled semen protocols is essential to increase pregnancy rates and reproductive efficiency in donkeys. This study aimed to evaluate the effect on sperm kinetic parameters and membrane integrity in cooled donkey semen diluted with defined milk proteins extender with 1% or 2% of egg yolk and the removal of seminal plasma. Twenty-four ejaculates from six jackasses were collected. Each ejaculate was divided into four aliquots that were diluted in extender with 1% (EY1) or 2% (EY2) egg yolk. One sample from each group was centrifuged, seminal plasma was removed (CEY1, CEY2 groups, respectively), and the samples were then refrigerated at 5 °C for 24 h. Fresh and cooled semen samples were assessed for sperm motility, morphology, and plasma membrane integrity. Total motility, progressive motility, sperm kinetic parameters, or live sperm cells were not statistically different when semen was cooled with an extender supplemented with 1% or 2% of egg yolk. Seminal plasma removal does not affect total motility or sperm kinetic parameters. However, progressive motility decreased (P<0.05) when semen was extended with 2% of egg yolk and seminal plasma was removed. Membrane integrity was affected (P<0.05) in centrifuged samples. In conclusion, the obtained results suggest that there is no difference in sperm kinetics and membrane integrity when 1% or 2% of egg yolk was added to the Equiplus extender. Also, the removal of seminal plasma by centrifugation did not have any beneficial effect on cooled donkey semen. Further studies are needed to relate these results with in vivo fertility tests with cooled donkey semen.(AU)

O desenvolvimento de protocolos de sêmen resfriado eficazes é essencial para aumentar as taxas de prenhez e eficiência reprodutiva em jumentos. O objetivo desse estudo foi avaliar o efeito do diluente à base de proteínas do leite com 1 ou 2% de gema de ovo sobre os parâmetros cinéticos do sêmen e integridade da membrana em sêmen resfriado de jumento, com ou sem a remoção do plasma seminal. Vinte e quatro ejaculados de seis jumentos foram coletados. Cada ejaculado foi dividido em quatro alíquotas e diluído em diluente com 1% (EY1) ou 2% (EY2) de gema de ovo. Uma amostra por grupo foi centrifugada e o plasma seminal removido (grupos CEY1 e CEY2, respectivamente). Os pellets foram novamente ressuspendidos nas mesmas concentrações e diluentes. Em seguida, as quatro alíquotas foram refrigeradas a 5°C por 24 horas. Amostras de sêmen fresco e refrigerado foram avaliadas quanto à motilidade espermática e integridade da membrana plasmática. Motilidade total, motilidade progressiva, parâmetros de cinética espermática ou células espermáticas vivas não apresentaram diferença significativa quando o sêmen foi resfriado com diluente suplementado com 1% ou 2% de gema de ovo. A remoção do plasma seminal não afetou a motilidade total ou os parâmetros de cinética espermática; entretanto, a motilidade progressiva diminuiu (P<0,05) quando o sêmen foi diluído com 2% de gema de ovo e o plasma seminal removido. Nas amostras centrifugadas, a integridade da membrana foi afetada (P<0,05). Em conclusão, os resultados sugerem que não há diferença na cinética espermática e na integridade da membrana quando 1% ou 2% de gema de ovo são adicionados ao diluente Equiplus e a remoção do plasma seminal por centrifugação não teve nenhum efeito benéfico no resfriamento de sêmen de jumento. Mais estudos são necessários para relacionar esses resultados com testes de fertilidade in vivo com sêmen resfriado em jumentos.(AU)

Fractionated semen collection as a tool to rescue fertility in stallions with seminal vesiculitis.

Treatments for seminal vesiculitis have poor outcomes in stallions; thus, the development of alternative strategies is warranted. This study aimed to evaluate fractionated semen collection as a method to restore the fertility of stallions diagnosed with seminal vesiculitis. Eighteen ejaculates from six stallions (three ejaculates/stallion) diagnosed with seminal vesiculitis were harvested in fractions, as follows: Fraction A (FA), the first two jets; Fraction B (FB), the third and fourth jets; and Fraction C (FC), the fifth and remaining jets of the ejaculate. All fractions were subject to standard semen evaluations that were performed in addition to cytology and bacterial aerobic cultures. Fractions were extended and cooled to 5 °C. As a proof of concept, 20 mares (48 estrous cycles, ∼8 cycles/stallion) were bred with 1 billion sperm from FA (cooled at 5 °C for 24 h). In our study, FA had negative bacterial cultures, absent macroscopic or microscopic abnormalities; FB had positive bacterial cultures in two stallions and presence of polymorphonuclear neutrophils (PMNs) in all samples, but with no macroscopic abnormalities; and FC had positive bacterial cultures, purulent appearance, and the presence of degenerated PMNs, just as noted in the whole semen. Overall, post-cooling sperm motility results were superior (P < 0.05) for FA in comparison with FB and FC. First cycle pregnancy rates using FA varied from 66% to 86%. None of the non-pregnant mares developed endometritis. In conclusion, fractionated semen collection can be used to obtain semen free of contamination and to achieve satisfactory pregnancy rates from stallions with seminal vesiculitis.

Cervical artificial insemination in sheep: sperm volume and concentration using an antiretrograde flow device.

There has been development of an antiretrograde flow device (DARIO), for sheep cervical artificial insemination (CAI). There, however, needs to be optimization of sperm volume and concentration of insemination doses when the DARIO is used for CAI. Objectives were to compare fertility rates (proportion of ewes lambing as a result of CAI) when there was use of the DARIO for CAI: two sperm volumes containing equal numbers of spermatozoa: 0.25 mL of 1,600 × 106 spermatozoa/mL and 0.50 mL of 800 × 106 spermatozoa/mL (Test 1 group), and two sperm volumes with a different number of spermatozoa/AI dose: 0.25 mL and 0.50 mL of 1,600 × 106 spermatozoa/mL (Test 2 group). There were 335 ewes from seven farms assigned to 60 batches (equally divided into a Control and Test 1 group). For the Test 2 group, 462 ewes from nine farms were assigned to 88 batches (equally proportioned into Control group and Test 2 groups). For the Test 1 group, proportion of ewes lambing as a result of CAI were 0.701 ±â€¯0.2679 and 0.595 ±â€¯0.2393 for the Control and Test 1 groups, respectively (P = 0.163). For the Test 2 group, proportions of ewes lambing were 0.550 ±â€¯0.2598 and 0.658 ±â€¯0.2412 for the Control and Test 2 group, respectively (P = 0.041). An inclusion of a larger number of spermatozoa per insemination in a 0.50 mL dose volume resulted improved proportion of ewes lambing as a result of CAI when there was used of the DARIO.

Effects of L-Carnitine on Equine Semen Quality During Liquid Storage.

l-Carnitine (LC) plays a key role in sperm metabolism, easily providing energy through ß-oxidation, which positively affects motility. The objective of this study was to investigate the association between blood plasma and seminal plasma LC levels, as well as the effect of LC as an additive in a skimmed milk-based extender during sperm storage at 5°C. In the first experiment, semen and blood samples from 14 Quarter Horse stallions were used. The LC content in blood plasma and seminal plasma was determined by spectrophotometry and their relationships with seminal parameters were evaluated. In the second experiment, ejaculates (n = 16) from four Quarter Horses were used. Each ejaculate was split into four treatment groups with different LC concentrations: 0 (control), 0.5, 1.0, and 2.0 mM. Sperm motility, integrity of plasma and acrosomal membranes, intracellular reactive oxygen species content, and plasma membrane stability were evaluated immediately after samples reached 5°C (0 hour) and after 24, 48, and 72 hours. There was a positive correlation (p < 0.05) between LC levels in seminal plasma with both sperm concentration and plasma and acrosomal membrane integrity. Furthermore, the addition of LC (1 and 2 mM) preserved the motility of equine sperm stored at 5°C. It was concluded that the concentrations of LC with seminal plasma present correlate to semen parameters and the addition of LC to skimmed milk-based extender preserves the motility of equine sperm stored at 5°C for up to 48 hours.

Effect of sperm dosage transportation in stallions: Effect on sperm DNA fragmentation.

Artificial insemination programs for horses usually involve ex vivo handling and transporting of sperm. The present experiment was designed to: (i) assess the effect of transportation on sperm DNA integrity at different time post semen collection, and (ii) evaluate if sperm DNA quality deteriorates rapidly beyond 24 h of cooled storage. After collection, the ejaculates were extended using INRA 96 and semen was prepared for prompt analysis (A0) or 24 h/48 h cooled-shipping (B24 and C48 respectively). Each sample was assessed for sperm DNA fragmentation index (SDFI) at time 0 and after incubation for 2, 6 and 24 h at 37 °C. There was very little difference in SDFI between freshly extended (A0) and 24 h/48 h cooled-transported semen samples (B24/C48) at time 0. After 2 h of incubation at 37 °C, there was an increase in SDFI ranging from 2.7% to 7.5% per hour in freshly extended semen samples (A0: 5.1 ± 1.5), while cooled-transported semen samples had a much greater increase in SDFI, ranging from 5.0% to 20.5% (B24: 14.7 ± 5.6) and from 8.2% to 26.8% (C48: 18.3 ± 7.2) respectively. There were not marked differences in the sperm DNA integrity between 24 and 48 h for transported samples, thus there is the possibility of desirable fertility with use of stallion sperm after 48 h of cooled storage.

Effects of ascorbic acid 2-glucoside and alpha-tocopherol on the characteristics of equine spermatozoa stored at 5°C.

The aim of this experiment was to evaluate the effects of adding ascorbic acid 2-glucoside (AA2G), a water-soluble antioxidant and stable derivative of ascorbate, to the semen extender and compare it to the addition of vitamin C (Vit. C) and the fat-soluble antioxidant α-tocopherol (α-Toh), both individually and in combination, on the seminal variables of equine sperm submitted to cooling for 72 h. We used two ejaculates from 10 stallions and evaluated them for motility, membrane integrity, chromatin fragmentation, mitochondrial activity and lipid peroxidation. In the analysis of lipid peroxidation, the control group showed 2506.2 ± 796.4 ng malondialdehyde/108 sperm, which was higher (P < 0.05) than the groups treated with antioxidants. The average value of motility in the AA2G group was 68.4 ± 18.1%, which was higher (P < 0.05) than that observed in the control group (62.1 ± 16.2%). The variables membrane integrity, chromatin fragmentation and mitochondrial activity did not show significant difference (P > 0.05) between treatments. It was concluded that the antioxidants protected sperm cells from lipid peroxidation and that AA2G was effective during the cooling process of equine semen at 5°C for72 h, providing increased levels of total motility.

Influences of dietary supplementation with Lepidium meyenii (Maca) on stallion sperm production and on preservation of sperm quality during storage at 5 °C.

Stallion semen is damaged by oxidative stress during cooling and transport. Semen processing and extenders have been tested to improve the fertilizing capacity of semen and to preserve semen during transport. Dietary supplementation with natural antioxidants has been proposed to prevent oxidative damages. In this study, for the first time, the effect of dietary supplementation with Lepidium meyenii (Maca) on the characteristics of fresh and chilled stallion semen was evaluated. Maca is a traditional Andean crop used as a nutraceutical for the fertility-enhancing properties that are linked with antioxidant activity. The diet of five stallions was supplemented with 20 g of Maca powder daily for a total of 60 days. A control group of five stallions received the same diet without Maca. Semen was collected once before the administration of Maca (D0), twice during the administration at 30 and 60 days (D30 and D60), and finally twice at 30 and 60 days after the end of the administration (D90 and D120). Ejaculates were processed for cooled shipping at 5 °C and evaluated in the laboratory for total and progressive motility, acrosome integrity, and lipid peroxidation after collection and after 24, 48, and 72 h of storage. Dietary supplementation with Maca improved sperm concentration (from 213 ± 80.4 to 447 ± 73.1 × 106 spz/mL) and total sperm count (from 10,880 ± 4377 to 24,783 ± 4419 × 106 spz). The beneficial effects of Maca supplementation on motility and acrosome integrity in the raw semen were detected from the end of treatment with Maca (D60) until the end of the study (D120). Furthermore, during cooling storage, total motility, progressive motility, and acrosome integrity declined more slowly in the Maca-treated group than in the control group. Lipid peroxidation did not change during cooling storage in either group and did not show a significant difference between the two groups. In this study, the dietary supplementation with Maca increased sperm production and stabilized semen quality during chilled storage.

Physical characteristics and fertility of fractionated donkey semen cooled at 5°C / Características físicas e fertilidade do sêmen asinino fracionado e resfriado a 5ºC

The aim of this study was to evaluate the effect of two different extenders (Skimmed Milk Glucose - SMG or Lactose - Egg Yolk - LEY) on physical characteristics and fertility of fractionated donkey semen cooled at 5°C. For this, four Pêga donkeys were used as semen donors. The sperm rich fraction of the ejaculate was diluted preparing insemination doses containing 400 x 106 motile spermatozoa in a volume of 22 mL, cooled to 5°C and stored up to 48 hours in a container proposed by Palhares (1997). Sperm motility and vigor were assessed in fresh semen, after first semen dilution, before insemination, at 24 and 48 hours after storage. For the fertility evaluation, 44 mares were inseminated with semen stored for a period between 12 and 24 hours. The mares were inseminated on fixed days (Mondays, Wednesdays, and Fridays) after the detection of a follicle greater than a 30mm diameter in one of the ovaries through ovulation. Pregnancy diagnosis was performed on day 12 post-ovulation, using transrectal ultrasonography. Semen diluted in SMG showed superior sperm motility than LEY, at the Pre-AI evaluation (P<0.05). At 48 hours of storage, all donkeys had motility values between 45 and 53% for semen diluted in SMG, while only one donkey showed motility greater than 30% in the LEY treatment. The pregnancy rate/cycle for mares inseminated with semen diluted in SMG was superior than that obtained using LEY (56.52% vs 4.76%, respectively).(AU)

Objetivou-se com o presente experimento avaliar o efeito de dois diferentes diluidores (leite em pó desnatado glicose - SMG ou lactose gema de ovo - LEY) sobre as características físicas e a fertilidade do sêmen asinino coletado de forma fracionada e resfriado a 5ºC. Para isso, quatro jumentos da raça Pêga foram utilizados como doadores de sêmen. A fração espermática rica do ejaculado foi diluída preparando-se doses inseminantes contendo 400 x 106 espermatozoides móveis em um volume de 22 mL, resfriadas a 5ºC e armazenadas por até 48 horas em contêiner proposto por Palhares (1997). A motilidade e o vigor espermáticos foram avaliados no sêmen fresco, após a pré-diluição, antes das inseminações, às 24 e 48 horas de armazenamento. Para avaliação de fertilidade, 44 éguas foram inseminadas com sêmen armazenado por um período entre 12 e 24 horas, em dias fixos (segundas, quartas e sextas-feiras), após a detecção de um folículo de diâmetro maior ou igual a 30mm em um dos ovários, até a ovulação. O diagnóstico de gestação foi realizado a partir de 12 dias após a ovulação, por meio de ultrassonografia transretal. O sêmen diluído em SMG apresentou motilidade espermática superior à do LEY, já a partir do tempo pré-IA. Às 48 horas de armazenamento, todos os jumentos apresentaram valores de motilidade entre 45% e 53%, quando o sêmen foi diluído em SMG, enquanto apenas um jumento apresentou motilidade superior a 30% no tratamento utilizando LEY. A taxa de concepção/ciclo das éguas inseminadas também foi superior para o sêmen diluído em SMG em relação ao diluído em LEY (56,52% versus 4,76%, respectivamente).(AU)

Comparison of cushioned centrifugation and SpermFilter filtration on longevity and morphology of cooled-stored equine semen.

This study compares two methods for seminal plasma removal by evaluating sperm recovery rates, and motility and morphology of cooled-stored semen. Ejaculates were divided into three groups: control, filtration and cushioned centrifugation. Semen was extended to 25 million sperm/ml using a skim-milk-based extender and stored at 5°C for all groups. Sperm motility (total motility (%TM) and progressive motility (%PM)) was determined at 0, 24, 48 and 72 hours by a computer-assisted sperm analyser. Sperm morphology was assessed using differential interference microscopy. Overall, %TM of the centrifugation group was significantly higher than the filter group, but not significantly different than the control. No significant difference in %TM or %PM was detected for the control group and filter. Cushioned centrifugation was a superior method to obtain progressively motile sperm compared with control (P=0.03) and filter groups (P<0.001). No significant difference was found for the per cent of normal sperm cells and detached heads between the groups. This study demonstrated that cushioned centrifugation was a superior method to remove seminal plasma while preserving %TM and enhancing %PM for stallions under cooled storage over three days. However, as the differences appear to be negligible, the SpermFilter may represent an alternative for farms lacking a centrifuge.

Effect of month of conception on fertility of mares inseminated with jackass semen / Influência do mês da concepção sobre a eficiência reprodutiva de éguas inseminadas com sêmen asinino

Fertility obtained by cross-breeding mares (Equus caballus) with jackasses (Equus asinus) was evaluated. Two extenders, containing skim milk-glucose or egg yolk-glycine were used to study the fertility of mares inseminated with diluted jackass semen (T1 and T2) or diluted and cooled semen at 5°C for 12 hours (T3 and T4). A total of 272 cycles of 208 mares of undefined breeds were evaluated, being uniformly distributed between groups. The cycles were controlled by transrectal palpation and teasing, and mares were inseminated every Tuesday, Thursday and Saturday (three times/week), from the detection of a follicle with 3.0 to 3.5cm diameter in one of the ovaries until ovulation. Pregnancy was detected using transrectal palpation, teasing and ultrasound exams every 14 days. The extenders had no effect on fertility (P>0.05). Pregnancy rates for the first cycle were 64.52%, 61.11%, 50.72% and 54.17% and pregnancy rates/cycle were 63.64%, 54.55%, 52.69% and 47.06%, respectively, for T1, T2, T3 and T4. Differences in pregnancy loss rates between groups and effect of month of conception on fertility were found. Pregnancy loss rates were significantly higher (P<0.05) in January (38.46%) and in February and March (52.38%), with an average of 33.09%. The results indicate that mares conceiving at the end of the physiological reproduction time, carrying a mule embryo, are more susceptible to pregnancy loss.

Avaliou-se a eficiência reprodutiva de cruzamentos interespécies entre fêmeas equinas e reprodutores asininos da raça Pêga. Para tal, estudou-se o efeito de dois diluidores, à base de leite em pó desnatado-glicose ou glicina-gema de ovo, sobre a fertilidade de éguas inseminadas com sêmen fresco diluído (T1 e T2) ou diluído e resfriado a 5°C por 12 horas (T3 e T4). Foram utilizados 272 ciclos de 208 éguas mestiças, distribuídas uniformemente entre os tratamentos, após agrupamento por idade e categoria reprodutiva. Os ciclos foram acompanhados por palpação retal e rufiação, sendo as inseminações realizadas às terças, quintas e aos sábados (três vezes por semana), a partir da detecção de um folículo de 3,0 a 3,5cm de diâmetro, em um dos ovários, até a ovulação. As gestações foram acompanhadas por palpação transretal, rufiação e por ultrassonografia, realizada a cada 14 dias. As taxas de gestação obtidas, ao primeiro ciclo, não diferiram (P>0,05) entre os tratamentos, sendo de 64,52% para T1, 61,11% para T2, 50,72% para T3 e 54,17% para T4. Da mesma maneira, as taxas de gestação por ciclo também não foram influenciadas (P>0,05) pelos tratamentos, com valores de 63,64%, 54,55%, 52,69% e 47,06% para T1, T2, T3, e T4, respectivamente. No que diz respeito às taxas de perdas gestacionais, obtidas por meio das taxas de perdas e de perdas ajustadas, houve influência (P<0,05) dos tratamentos. Observou-se, ainda, efeito do mês da concepção, com taxas de perda superiores para os meses de janeiro (38,46%) e de fevereiro e março (52,38%), sendo a média de 33,09%. Sugere-se que a redução dos padrões de secreção do LH, ao final da estação de monta, responda pelas maiores taxas de perdas gestacionais do cruzamento interespécie, notadamente naquelas éguas que conceberam no final da estação de monta fisiológica dos equinos.

Efeito da categoria reprodutiva sobre a fertilidade de éguas inseminadas com sêmen asinino diluído e resfriado a 5ºC por 12 horas de armazenamento / Effect of mare status on fertility of inseminated mares with jackass semen diluted and cooled at 5ºC for 12 hours

Estudou-se o efeito da categoria reprodutiva sobre a fertilidade de éguas inseminadas com sêmen asinino diluído, resfriado e armazenado. Os ciclos foram acompanhados por palpação retal e rufiação, sendo as inseminações realizadas às terças, quintas e sábados, a partir da detecção de um folículo de 3,0 a 3,5cm de diâmetro, em um dos ovários, até a ovulação. O sêmen de cinco jumentos da raça Pêga foi diluído nos diluidores de leite em pó desnatado-glicose ou glicina-gema de ovo, resfriado a 5ºC e armazenado por 12 horas, sendo a dose inseminante de 400 x 106 espermatozoides móveis (no momento da diluição final, pré-resfriamento) depositada no corpo do útero. O diagnóstico de gestação foi realizado por meio de palpação transretal, rufiação e ultrassonografia, realizada a cada 14 dias. Os resultados de 195 ciclos, referentes a 141 éguas, foram agrupados de acordo com a categoria reprodutiva a que pertenciam: potra, égua solteira, égua parida e no "cio do potro". As taxas de concepção, ao primeiro ciclo, foram de 60,00%, 48,28%, 75,00% e 47,17% e, após quatro ciclos, de 61,54%, 47,13%, 54,76% e 47,17%, na mesma ordem para as categorias descritas anteriormente. A categoria reprodutiva não teve efeito (P>0,05) sobre a fertilidade das éguas inseminadas com sêmen asinino resfriado, sendo as potras, éguas solteiras, éguas paridas e no "cio do potro" igualmente eficientes para o uso na reprodução...

The effect of the mare status on fertility of mares inseminated with diluted, cooled and stored jackass semen, was studied. The cycles were controlled by transrectal palpation and teasing, and mares were inseminated every Tuesday, Thursday and Saturday, from the detection of a follicle with 3.0 to 3.5cm diameter in one of the ovaries until ovulation. The semen of five Pêga jackasses was diluted in skim milk-glucose or in egg yolk-glycine extender and cooled at 5ºC for 12 hours, with the inseminate dose of 400 x 106 motile spermatozoa (at the moment of the final dilution, before cooling). The inseminations were carried out in the uterine body. Pregnancy was detected using transrectal palpation, teasing and ultrasound exams every 14 days. The results of 195 cycles of 141 mares were grouped according to the mare status: maiden, barren, lactation or in foal heat. Pregnancy rates for the first cycle were 60.00%, 48.28%, 75.00% and 47.17%, and after four cycles, the pregnancy rates/cycle were 61.54%, 47.13%, 54.76% and 47.17%, respectively for maiden, barren, lactation and in foal heat mares (P>0,05). The mare status did not affect pregnancy rates of mares inseminated with diluted and cooled jackass semen and were efficient to use on reproduction...