The effects of equine somatotropin on pituitary and testicular function in the stallion during the nonbreeding season

An experiment was conducted to determine the effects of equine somatotropin on the reproductive axis of the stallion during the nonbreeding season. Adult stallions were treated with equine somatotropin (20 μg/kg body weight [BW]; n = 5) or saline (n = 4) daily for 21 days starting in January. During the last week of treatment, stallions were subjected to low- and high-dose injections of luteinizing hormone (LH), as well as low- and high-dose injections of gonadotropin-releasing hormone (GnRH) and thyrotropin-releasing hormone (TRH). Two months after the onset of somatotropin treatment, semen was collected from all stallions every other day for 14 days. Treatment with equine somatotropin increased (P < .001) daily IGF-1 concentrations but had no effect (P > .1) on concentrations of LH, follicle-stimulating hormone (FSH), or testosterone. The testosterone responses to injections of LH were similar (P > .1) between treatments. Likewise, the LH, FSH, prolactin, and testosterone responses to the injections of GnRH/TRH were similar (P > .1) between groups. At seminal collections, stallions treated with somatotropin exhibited greater volumes of gel-free semen (P < .01) and gel (P < .05) and had decreased time until ejaculation (P < .05). In conclusion, somatotropin treatment for 21 days may alter the long-term accessory gland contribution to seminal volume but does not appear to alter pituitary gonadotrope function or testicular testosterone secretion.     

Sodium Caseinate and Cholesterol Improve Bad Cooler Stallion Fertility

This study aimed to assess the effects of sodium caseinate and cholesterol to extenders used for stallion semen cooling. Two ejaculates from 19 stallions were extended to 50 million/mL in four different extenders and cooled-stored for 24 hours at 5°C. The extender 1 (E1) consisted of a commercially available skim milk–based extender. The extender 2 (E2) consisted of E1 basic formula with the milk component being replaced by sodium caseinate (20 g/L). The extender 3 (E3) consisted of E1 basic formula added to cholesterol (1.5 mg/120 million sperm). The extender 4 (E4) consisted of a combination of the E2 added to cholesterol. At 24 hours after cooling, sperm motility parameters, plasma membrane stability (PMS), and mitochondrial membrane potential were assessed. In addition, cooled semen (1 billion sperm at 5°C/24 hours) from one “bad cooler” and one “good cooler” stallions, split into four extenders was used to inseminate 30 light breed mares (30 estrous cycles/extender). Milk-based extenders (E1 and E2) had superior sperm kinetics than E3 and E4 (P < .05). Plasma membrane stabilization was significantly higher (P < .05) in E4 than E1, whereas E2 and E3 presented intermediate values (P > .05). The mitochondrial potential intensity was lower (P < .05) in E2 and E4 groups compared with E1 and E3. The good cooler stallion had high fertility (∼80%) in all extenders. However, for bad cooler stallion, E1 40% (8/20) and E2 45% (9/20) had poor fertility (P < .05) compared with E4 85% (17/20), whereas E3 55% (11/20) had intermediate value (P > .05). In conclusion, the association of sodium caseinate and cholesterol improved fertility of bad cooler stallion semen cooled for 24 hours.     

Cryopreservation of Cooled Semen and Evaluation of Sperm Holding Media for Potential Use in Equine-Assisted Reproduction Procedures

Processing stallion semen for assisted reproductive procedures, such as intracytoplasmic sperm injection (ICSI), requires special considerations regarding cooling, concentrating, and handling of sperm. The aim of experiment 1 was to determine whether cooled semen could be frozen without removal of seminal plasma and at a low sperm concentration while maintaining motile sperm for ICSI selection procedures. In experiment 2, five media for holding stallion sperm were compared to evaluate sperm motility for an interval of time sufficient for ICSI sperm selection procedures. In experiment 1, semen samples from eight stallions were cooled for 24 hours in two extenders, CST (E-Z Mixin-CST “Cool-Store/Transport” Animal Reproduction Systems) and INRA96 (Institut National de la Recherche Agronomique, IMV International Corporation), before being frozen in four freezing diluents, and were evaluated at 0, 45, and 75 minutes after thawing. The cooling extender did not significantly affect sperm motility, but modified French and glycerol egg yolk diluents provided the best sperm motility for frozen–thawed groups. In experiment 2, semen samples from seven stallions were used to test five media for holding sperm. Samples were analyzed for total and progressive motility at hourly intervals. Mean total and progressive motility were not different (P > .05) among groups from 1 through 4 hours. At 5 hours, groups differed (P = .004), with sperm held in Tyrode’s with albumin, lactate, and pyruvate having higher (P < .05) total and progressive motility than all other samples. In conclusion, motile stallion sperm can be obtained after the sperm are cooled for 24 hours, frozen, and thawed; various media are available to maintain sperm motility during equine ICSI selection procedures.     

Fertility of Mares Inseminated With Frozen-Thawed Semen Processed by Single Layer Centrifugation Through a Colloid

The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by single layer centrifugation (SLC) through a colloid before insemination. In addition, changes in semen parameters, including motility, were determined before and after SLC. Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- × 0.5-mL straws (200 × 10⁶/mL) of frozen-thawed semen. Treatment mares were inseminated with 4 × 0.5 mL of frozen-thawed semen after processing by SLC. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test. The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen, despite the difference in sperm number (171 × 10⁶ ± 21, 59 × 10⁶ ± 25 progressively motile sperm). After frozen-thawed semen was processed by SLC, the percentage progressively motile sperm improved (P < .05), and SLC processing resulted in a 21.8% recovery of spermatozoa. In summary, centrifugation of frozen-thawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination.     

Effect of Sire-Associated Factors on Secondary Sex Ratio of Offspring in Equine

The objective of the present study was to evaluate the effect of breed of stallion and individual stallion on secondary sex ratio (SSR; the proportion of male foals at birth). Data associated with the sex of foals and the sire, as well as the breed and age of sire were retrieved from the database of the Equestrian Federation of the Islamic Republic of Iran. In total, data consisted of 4,491 birth records from 92 stallions. Stallions were from three breeds of Arabian, Thoroughbred, and Akhal-Teke. Data were analyzed using univariable and multivariable logistic regression. Proportion of colts was 63.0% (427/678), 46.1% (1,545/3,355), and 53.9% (247/458) in Arabian, Thoroughbred, and Akhal-Teke stallions, respectively. In Arabian stallions, SSR was skewed toward males (P < .0001; odds ratio, 1.701), whereas in Thoroughbred stallions, it was skewed toward females (P = .001; odds ratio, 0.853). Secondary sex ratio was not skewed in Akhal-Teke stallions (P > .05). Secondary sex ratio in Thoroughbred stallions was lower than that in Arabian (P < .0001; adjusted odds ratio, 1.983) and Akhal-Teke (P = .010; adjusted odds ratio, 1.527) stallions, but SSR did not differ between Arabian and Akhal-Teke stallions (P > .05). There was the effect of individual stallion on SSR in Arabian and Thoroughbred breeds (P < .0001) but not in Akhal-Teke breed (P > .05). In conclusion, the present study showed the effect of breed of stallion and stallion itself on sex ratio of foals.     

Dose-Response of Prolactin to Increasing Doses of the Dopamine Receptor Antagonist,Sulpiride, in Horses: Effect of Season in Mares and Stallions of Estradiol Pretreatment in Geldings

Two experiments were performed to determine whether dopaminergic input to the adenohypophysis (1) differs across seasons in mares and stallions proportionally with changes in prolactin secretion and (2) is altered by estradiol administration in geldings. In experiment 1, prolactin responses to increasing doses of l-sulpiride in eight mares and eight stallions in March, June, September, and December were used to estimate the theoretical dose equivalent to 50% of maximal response. Prolactin areas increased (P < .001) with increasing doses of sulpiride and were greatest (P < .05) in March for stallions, but in June for mares. Mean half-maximal dose, which was assumed to be proportional to the dopaminergic input to the pituitary, was lowest (P < .05) in June and greatest in September. Experiment 2 used the same approach to determine whether the stimulatory effect of estradiol pretreatment on prolactin secretion was associated with an alteration of the half-maximal response. Geldings (n = 6/group) were administered 100 mg of estradiol cypionate in oil, or oil alone, on day 0 (October 3) and increasing doses of l-sulpiride starting on day 6. Estradiol treatment increased (P < .08) the prolactin response to l-sulpiride at 0.41 μg/kg body weight and all higher doses (P < .05); mean half-maximal dose did not differ (P > .1) between groups. We conclude that dopaminergic input to the adenohypophysis of mares and stallions varies with season and that the stimulatory effect of estradiol on prolactin secretion is not associated with a decrease in dopaminergic input to the adenohypophysis.     

The Use of Cefquinome in Equine Semen Extender

Antibiotics are commonly used in equine semen extender for conservation, if semen has to be stored cooled for a maximum of 48 hours or frozen, to eliminate pathogenic or potentially pathogenic bacteria from semen and reduce the risk of postmating endometritis. Little is known about the effect of antibiotics on spermatozoa when semen is stored over a longer period. Cefquinome, a broad spectrum antibiotic and fourth-generation cephalosporin, has been proven to be a powerful drug for the treatment of endometritis and mastitis in different species. Recently in equine studies, it was found to localize in high concentrations in the endometrium. Therefore, cefquinome was used as the antibiotic in semen extender and compared with a commercial semen extender containing gentamicin for effects on motility and membrane integrity of spermatozoa. During the breeding season, ejaculates from nine light horse stallions were collected and half of each ejaculate was stored for 48 hours in modified Kenney type semen extender containing either cefquinome or gentamicin. At 0, 24, and 48 hours, aliquots (20 μL) of the stored semen were evaluated for (progressive) motility and membrane integrity, as well as for various motility parameters by computer assisted sperm analysis. No differences (P > .05) were found in total motility or progressive motility between extenders at any time point. However, there were differences (P < .05) in velocity parameters, although the effect of velocity parameters on fertility is not clear. In general, semen parameters after storage in non-fat dried skim milk semen extender containing cefquinome are comparable with those after storage in semen extender containing gentamicin. The wider spectrum of bactericidal activity possessed by cefquinome may prove to be beneficial in some cases.     

Effects of α-tocopherol and Ascorbic Acid on Equine Semen Quality after Cryopreservation

This study investigated the effects of ascorbic acid and α-tocopherol supplementation on semen quality parameters of equine thawed-frozen semen. Semen was divided in seven different treatments in a final concentration of 100 × 10⁶ sperm/mL by using Gent extender containing no supplements (control) and the following supplements with three different concentrations: α-tocopherol (0.5, 1, and 2 mM) and ascorbic acid (0.45, 0.9, and 1.8 g/L). After thawing, all samples were maintained at 37°C, while analyses were performed at 0, 60, and 120 minutes. Evaluation of viability and acrosome status (using Pisum sativum agglutinin conjugated to fluorescein isothiocyanate and propidium iodide), mitochondrial membrane potential (5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolyl carbocyanine iodine [JC-1]), membrane lipid peroxidation (LPO; C₁₁-BODIPY^581/591), and stability of the plasmatic membrane (merocyanine 540 and Yo-Pro-1) of each sample was determined by flow cytometry. Relative to the control group, supplementation with α-tocopherol improved (P ≤ .05) postthaw membrane LPO, yet the higher concentrations of ascorbic acid (0.9 and 1.8 g/L, respectively) showed a negative effect on membrane LPO. Neither antioxidant significantly increased (P > .05) the acrosome integrity and mitochondrial membrane potential of frozen-thawed spermatozoa, although supplementation with α-tocopherol and ascorbic acid (0.9 and 1.8 g/L, respectively) had a positive effect on membrane integrity and stability (P ≤ .05). For all semen parameters, the lower concentration of ascorbic acid (0.45 g/L) did not show significant differences (P > .05) compared with the control. In conclusion, α-tocopherol seems to be an efficient antioxidant for reducing the oxidative stress provoked by cryopreservation, decreasing lipid peroxidation on equine spermatozoa.     

Influence of Seminal Plasma Antioxidants and Osteopontin on Fertility of the Arabian Horse

This study was designed to investigate enzymatic antioxidants’ activity and nonenzymatic antioxidants’ levels in seminal plasma of stallions and to relate them with season, age, and fertility of stallions. Fifty ejaculates were collected from six healthy Arabian stallions, 4-22 years old. Ejaculates were evaluated by conventional methods. Five milliliters of each semen sample was centrifuged, and the supernatant seminal plasma was stored at −20°C. Five antioxidants, in addition to osteopontin (OPN) and testosterone, were determined in stallion seminal plasma by using commercial enzyme-linked immunosorbent assay kits. Results revealed that uric acid, ascorbic acid, OPN, and testosterone concentrations and glutathione peroxidase (GPx) activity in stallions’ seminal plasma were high (P < .05) during spring. GPx activity was higher (P < .05) in age group B (11-18 years old) than in age group A (4-10 years old). The effect of stallions’ age on GPx activity in the fertility groups was highly significant (P < .01). OPN concentration was highest (P < .05) in age group A. Uric acid and OPN concentrations and GPx activity in stallions’ seminal plasma and percent of sperm motility were higher (P < .05) in fertility group III (>70%) than in fertility group I (<50%). However, ascorbic acid concentration, catalase activity and percentage of sperm abnormalities were lower (P < .05) in fertility group III than in fertility group I. It was concluded that season and stallion age may affect antioxidant defense systems in stallions’ seminal plasma. The impairment of seminal antioxidants and OPN could lead to low fertility.     

The Effects of an Oxygen Scavenger and Coconut Water on Equine Sperm Cryopreservation

Alternative sources of lipoproteins in semen extenders could replace animal by-products. We hypothesized that: (1) post-thaw semen parameters and fertility would not be different in coconut water (CW)–treated samples compared with egg yolk (EY)–treated samples and (2) the use of an oxygen scavenger (Oxyrase) would improve post-thaw sperm motility and membrane integrity and decrease lipid peroxidation. Experiment 1: three ejaculates each from five stallions were split into four treatments: EY, CW, egg yolk with Oxyrase, and coconut water with Oxyrase. Computer-assisted sperm analysis measured progressive and total motility, velocity, and linearity. Membrane integrity, apoptosis, and lipid peroxidation were evaluated using propidium iodide, annexin, and BODIPY fluorescent probes, respectively. Samples were cryopreserved, stored in liquid nitrogen, and then thawed to 37°C and analyzed again. Experiment 2: one ejaculate was divided into two aliquots and cryopreserved using either CW or EY. In a crossover design, 12 mares were bred on two consecutive cycles with either EY or CW. Pregnancy evaluations were at 14-day gestation. No differences were detected in sperm parameters between CW and EY (P > .05). Oxyrase did not improve sperm motility parameters in post-thaw samples, nor did it show protective effects for viability or against membrane damage (P > .05). More mares became pregnant using CW than EY (11/12 vs. 6/12, respectively; P = .013). Use of CW is a viable alternative to animal-based products in the cryopreservation of stallion semen.     

Effect of Removing Seminal Plasma Using a Sperm Filter on the Viability of Refrigerated Stallion Semen

Cooling of equine semen obtained from some stallions results in lower seminal quality and viability when the seminal plasma (SP) is present. The objective of this study was to evaluate the effect of the removal of SP using a Sperm Filter on the viability of cooled stallion semen. For this purpose, 31 stallions were used. Their ejaculates were divided into three groups: CN, semen was diluted with an extender; FLT, SP was removed by filtration; and CT, SP was removed by centrifugation and cooled to 15°C for 24 hours. Sperm kinetics and plasma membrane integrity were evaluated immediately after collection (T0) and after 24 hours of refrigeration (T1). No difference (P > .05) was noted at T1 for total sperm motility (TM), progressive sperm motility, or plasma membrane integrity when semen samples from all the stallions were analyzed. However, when samples from stallions termed “bad coolers” were analyzed (TM = <30% at T1), a difference was observed in TM and progressive sperm motility for CN compared with FLT and CT at T1. Sperm recovery was greater when SP was removed using the filter (FLT) to that when the SP was removed by centrifugation (CN) (89% vs. 81%). Thus, we concluded that filtering with a Sperm Filter is an efficient and practical method for removal of SP from stallion ejaculates, with lower sperm loss than centrifugation. We also found that the presence of SP reduces the quality and viability of cooled semen from stallions whose semen is sensitive to the process of refrigeration.     

Effect of Marcha Exercise on Serum Electrolytes and Acid–Base Balance in Mangalarga Marchador Horses

The marcha competition is a functional evaluation, without similar in the world. In this competition, the animals perform marcha at average speed between 9 and 12 km/hr for 50 minutes, covering roughly 6 to 7 km with no rest. The aim of this study was to evaluate and compare the effect of batida gait competition on acid–base balance in Mangalarga Marchador horses during an official marcha competition. The study was conducted on 24 Mangalarga Marchador horses, 12 stallions, and 12 mares (aged 4–6 years and 399 ± 45 kg of body weight). Venous blood samples were collected before and after competition. Blood samples were analyzed for acid–base balance, as well as biochemical and electrolyte parameters. In stallions, a significant (P < .05) postexercise increase in pH, partial pressure of oxygen, bicarbonate concentration, base excess, and glucose was noted. Decrease (P < .05) in pCO₂ (partial pressure of carbon dioxide), chloride and ionized calcium were observed after exercise only in stallions. No changes (P > .05) in hematocrit, hemoglobin, sodium, and potassium were observed after exercise in all animals. The changes in the acid–base balance and electrolyte profile of the Mangalarga Marchador stallions after marcha competition resulted in hypochloremic metabolic alkalosis. Marcha competition did not cause acid–base disturbance in mares. This study enables an evaluation and comparison of physical effort caused by the marcha exercise on acid–base balance in Mangalarga Marchador horses and provides further evidence of the existence of gender differences in sports horses.     

Cryopreservation of Stallion Spermatozoa with INRA96 and Glycerol

The aim of the present study was to improve success of cryopreservation of stallion spermatozoa. Semen from eleven stallions was collected and frozen in INRA 96 with two different concentrations of glycerol (3.5% and 6.0%) and compared with a control freezing process. The mean post-thaw motility for the eleven stallions of 57.93% (3.5% glycerol) and 66.50% (6.0% glycerol), which was statistically higher (P < 0.05) when compared with the mean post-thaw motility (39.7%) for semen in a control egg-yolk extender (Equipro^® CryoGuard™ Complete, Minitube). The Equipro^® CryoGuard™ Complete is a commercial semen freezing protocol that has been one of the standard processes used in our laboratory for freezing equine spermatozoa. INRA 96 with 6% added glycerol was used in the fertility trial as it provided the highest spermatozoa survival. To evaluate fertility of the frozen semen, eight mares were bred over two cycles with both fresh and frozen semen. The pregnancy rate of mares bred with frozen semen (55.6%) was not statistically different (P > 0.05) from the pregnancy rate of mares bred with fresh semen (55.6%). INRA 96 with 6.0% glycerol improved the survivability of stallion spermatozoa through the cryopreservation process, and subsequent fertility was not different (P > 0.05) from fresh, extended semen.     

Effects of Diet and Weight Gain on Body Condition Scoring in Thoroughbred Geldings

In the horse, the body condition score (BCS) system to assess subcutaneous fat deposition is a useful tool for making feeding management decisions. The system includes assessing fat deposition at six body areas (neck, withers, shoulders, ribs, loin, and tail head) and was developed in Quarter Horse mares. It has not been tested for use on other breeds or genders, possibly compromising the system's ability to describe fat accretion in other classes of horse. The objective of this study was to examine the ability of the previously developed fat accretion characteristics to describe fat deposition in mature Thoroughbred (TB) geldings. An additional goal was to determine whether fewer body areas could be used. Fifteen mature TB geldings were fed for weight gain over an 8-month period on one of two diets and were independently assessed for BCS on a monthly basis by two judges. BCS was determined by averaging the scores that were assigned to each of the six body areas. Fat accretion characteristics were also recorded. Across both diets, the neck area scored significantly higher than the withers and loin (P < 0.05) throughout the study. A BCS derived only from the body areas of neck, shoulders, ribs, and tailhead was found to accurately predict the six body area−derived mean BCS. The results of this study provide justification for modifications of the BCS system for use in TB geldings and also demonstrated that fewer body areas can be used to accurately predict mean BCS.     

The relationships between scrotal surface temperature,age and sperm quality in stallions

In horses, spermatogenesis normally occurs at an average intratesticular temperature of 35 °C; therefore, mechanisms for testicular thermoregulation are essential. Measuring the scrotal surface temperature by thermography is one of the methodologies used to evaluate the effectiveness of testicular thermoregulation. The objective of this study was to determine the relationship between the control of scrotal surface temperature and sperm quality in horses of different ages. In total, 24 Quarter Horse stallions were divided into three groups: YS (young stallions), AS (adult stallions) and OS (old stallions). Initially, we calculated the testicular volume (TV) and evaluated various aspects of the semen (sperm kinetics, plasma membrane integrity and sperm morphology) for all the animals. We also evaluated rectal temperature (RT), body surface temperature (BST,) and average scrotal surface temperature in the testicular region (SST) before (M0) and after sun exposure (M1). Differences were observed (p<0.05) between the RT and BST before and after sun exposure in all three groups. However, there were no differences (p>0.05) in the SST values at these two time points, thus demonstrating the efficiency of the mechanisms for testicular thermoregulation. The SST was similar (p>0.05) among all three groups. Based on these results, we conclude that fertile stallions of different age groups are able to maintain SST and measuring the heat radiating from the scrotum using a digital infrared thermographer. We can also conclude that measuring the heat radiating from the scrotum using a digital infrared thermographer is a practical and efficient tool for monitoring SST in horses.     

Markers for Predicting Overweight or Obesity of Broodmares

Obesity has become of great concern to all equine community from both veterinary and welfare points of view. For estimating obesity markers of brood mares, 17 mares with body conditions were subjected to blood sampling and ultrasound examination to measure rump fat for 6 consecutive weeks. Body length (L), girth (G), and height (H) were measured to estimate body weight (BW), body fat %, body fat mass (BFM) and body mass index (BMI). Mares were classified into three groups according to body condition score (BCS) and rump fat thickness (RF). Overweight mares (O) had BCS >7 and RF >7 mm, moderate (M) had BCS and RF >3 to ≤7, and emaciated (E) had BCS and RF ≤ 3 mm. Glucose, triglycerides, nitric oxide (NO), insulin, insulin-like growth factor-I (IGF-1), leptin, ovarian hormones, and thyroid hormones were measured. Results revealed that BCS, G, L, L × G × H, BW, RF, fat %, and BFM correlated significantly (P < .0001) with body condition. Tetraiodothyronine concentrations of E mares were significantly high (P = .04), but triiodothyronine concentrations tended (P = .07) to be low. Insulin (P = .06) and IGF-1 (P = .07) concentrations tended to be high in O mares. Moderate mares had the highest leptin concentrations (P = .007), but E mares had the lowest P4 concentrations (P = .01). Overweight mares had nonsignificantly high glucose, NO, and triglycerides. In conclusion, back fat and morphometric measurements are the easiest and simple assessment of overweight and obesity. Obese and overweight mares showed slight hyperinsulinemia, hypertriglyceridemia, and hyperglycemia. Hyperleptinemia alone is not indicative of obesity.     

The onset of puberty of Pelibuey male hair sheep is not delayed by the short term consumption of Morus alba or Hibiscus rosa-sinensis foliage

The present trial evaluated the effect of phytoestrogens consumed from Mulberry (Morus alba) or Hibiscus (Hibiscus rosa-sinensis) foliage, on the onset of puberty of Pelibuey male lambs. The design of the study aimed at identifying possible acute changes on the onset of puberty. Eighteen male lambs were used (107±9 days of age, 16±3 cm scrotal circumference (SC), 3.4±0.1 body condition score (BCS) and 17±1 kg bodyweight (BW)). At weaning, the lambs were distributed in three groups (n=6 each): (i) Control group (balanced feed based on grains+star grass), (ii) Mulberry Group (Mulberry foliage+balanced feed) and (iii) Hibiscus Group (Hibiscus foliage+balanced feed). BW, SC and body condition score (BCS) were determined every 14 days. An estrogenized adult female was used to attempt the collection of semen samples from the males two times per week. The onset of puberty was determined as the presence of sperm showing progressive movement in the ejaculate. From then, semen collections continued in the males (once a week) until the first normal ejaculate was obtained. The nutritional value of the foliages, as well as the content of total phenols, total tannins and condensed tannins, was determined. The phytoestrogen content (Equol, 4′7-dimetoxyisoflavone, Genistein, Rutin and Ononin) was determined from the methanolic extract of both plants. Total dry matter consumptions were similar between the Control and Mulberry groups and both were higher than the Hibiscus group (P<0.05). The total consumption of phytoestrogens and tannins was similar in the Mulberry and the Hibiscus group. The short-term consumption of phytoestrogens from Mulberry or Hibiscus foliage failed to delay the onset of puberty. The mean age at the onset of puberty was similar in the three groups (146 days in the control and Mulberry groups and 154 days in the Hibiscus group; P>0.05). The mean age at the first normal ejaculate was also similar between groups (167 days for Mulberry and Hibiscus groups and 169 days in the control group; P>0.05). The BW (Control: 21.6±3.1; Mulberry: 20.7±1.9; Hibiscus: 19.8±2.8 kg), the BCS and the SC were similar between groups at the onset of puberty. The present trial showed that a short exposure to M. Alba or H. rosa-sinensis foliage through diet did not influence the onset of puberty of Pelibuey male lambs.     

Quality of Raw and of Cold-Stored Semen in Icelandic Stallions

The aim of the present study was to evaluate the quality of raw and cooled semen in Icelandic stallions. Experiments were performed using seven stallions aged between 3 and 19 years. From each stallion, six ejaculates were collected, and semen quality was determined. Thereafter, the semen was split into eight equal parts and processed with and without centrifugation using the extenders INRA 82-egg yolk, INRA 96, GENT, and Equi-Pro to a final concentration of 30 × 10⁶ sperm/mL. The extended semen was then cooled in an Equitainer, where it was stored for 24 hours, and subsequently refrigerated for another 24 hours at 5°C. Immediately after dilution as well as after 24 and 48 hours storage, sperm motility was analyzed using computer-assisted sperm analyzer, and viability was assessed after dual DNA staining with SYBR-14 in combination with propidium iodide. The results show that the stallion had a significant (P < .05) influence on all variables evaluated in raw semen, and mean (±SEM) values of 43.4 ± 4.3 mL for the volume, 193.0 ± 17.0 × 10⁶ sperm/mL for the concentration, 6.7 ± 0.5 × 10⁹ for total sperm and 73.5 ± 2.1% for total sperm motility, 48.7 ± 2.0% for progressive motility, and 65.3 ± 2.0% for rapid cells were measured. In the cold-stored semen, all variables were significantly (P < .05) influenced by the stallion, extender, and storage time (48 hours). Except for Equi-Pro, all extenders examined were suitable for cooled semen preservation. For storage of more than 24 hours, centrifugation and removal of the seminal plasma were advantageous for all extenders with the exception of Equi-Pro.     

Quality of Fresh,Cooled, and Frozen Semen From Stallions Supplemented with Antioxidants and Fatty Acids

This study assessed the effect of oral supplementation with the primary antioxidants and fatty acids involved in spermatogenesis (L-carnitine, selenium, vitamin E, omega-3, and omega-6) on the seminal quality in fresh, cooled, and frozen semen of stallions (n = 8), using a randomized design. The animals were divided into Group I (n = 4) and Group II (n = 4) for a 30-week experiment. The two groups alternated between nutraceutical supplementation and a placebo over the course of the experiment. Semen collections were performed in two sets: once in the middle of the experiment, before the two groups switched treatments, and once at the end. The volume, appearance, sperm concentration, spermatozoa kinetics, and membrane integrity of fresh semen were evaluated. The spermatozoa kinetics and membrane integrity of cooled (for 24, 36, and 48 hours) and frozen semen were also evaluated. No differences were observed in volume, appearance, and sperm concentration between treatment and control. However, compared with placebo, nutraceutical supplementation increased (P < .05) total motility, trajectory speed, as well as plasma and acrosomal membrane integrity in spermatozoa from fresh semen. In cooled semen, nutraceutical treatment also increased (P < .05) total motility, speed, and membrane integrity of spermatozoa compared with the control. In frozen semen, supplementation increased (P < .05) spermatozoa progressive motility and plasma membrane integrity. Our results suggest a positive, synergistic effect of the antioxidant L-carnitine and selenium on spermatozoa kinetics. Similarly, the increase in plasma and acrosomal membrane integrity could be attributed to higher concentrations of polyunsaturated fatty acids (a key cell-membrane component), combined with the prevention of excess lipid peroxidation by antioxidants. In conclusion, supplementation with nutraceuticals containing fatty acids and antioxidants improved the quality of fresh, cooled, and frozen stallion semen. Therefore, nutraceutical use should increase the success of artificial insemination with cooled and cryopreserved semen.     

An approach to rescue the fertility of stallions with a high level of hemospermia

A high amount of blood and not the mere presence of blood in equine semen impacts fertility. The aim of this study was to develop an approach to rescue the fertility of stallions with high hemospermia levels. Semen from 15 stallions was divided into four experimental groups: (a) Control—pure raw semen, (b) WB50—50% (v/v) whole blood added into semen, (c) E1—WB50 extended in a 1:1 (v/v) ratio with milk-based extender and (d) E2—WB50 extended in a 2:1 ratio with milk-based extender. Sperm kinetics, plasma membrane integrity (PMI), lipid peroxidation (PER) and intracellular superoxide (O₂) production were immediately evaluated. Four cycles of 20 mares were randomly assigned to the experimental groups. Mares were bred with an insemination dose of 1 billion total sperm and pregnancy was diagnosed 14 days after ovulation. Sperm kinetics could not be evaluated in the WB50 samples. Total motility was lower (p < .05) in E1 than in CT and E2 samples. Progressive motility decreased (p < .05) with an increase in the percentage of blood in the samples. The PMI and PER did not differ between groups (p > .05); however, O₂ production was higher (p < .05) in WB50 than in E2 samples, while the values were intermediate (p > .05) for CT and E1 samples. The control (90%) and E2 (90%) groups had superior (p < .05) fertility than the others (WB50—0% and E1—25%). It was concluded that sperm motility and fertility of semen with a large amount of blood can be rescued by dilution with a 2:1 extender:semen ratio using a milk-based extender.