Testicular Measurements and Daily Sperm Output of Tori and Estonian Breed Stallions

Evaluation of testicular measurements and daily sperm output (DSO) yields valuable information for predicting the reproductive capacity of stallions. The present study evaluated testicular measurements (height, length, width and circumference) and DSO of eight Tori and eight Estonian breed stallions. One ejaculate of semen was collected daily for 10 subsequent days from each stallion. The gel‐free volume of semen was measured with a graduated glass cylinder and the sperm concentration was assessed with a Chorjajev chamber. The volume of gel‐free fraction was multiplied by the sperm concentration to give the total number of spermatozoa (TSN). The DSO was calculated as mean TSN of collection on days 8–10 in Tori breed stallions and on days 4–10 in Estonian breed stallions. The DSO of Tori breed stallions was 12.9 × 10⁹ spermatozoa and of Estonian breed stallions 4.5 × 10⁹ spermatozoa (p < 0.001). Testicular measurements (in cm) 1 day after the last semen collection were as follows: left testis– height 7.3, length 10.4 and width 7.3 in Tori breed stallions, and 5.9, 8.1 and 5.9, respectively, in Estonian breed stallions; right testis– height 7.4, length 10.6 and width 7.4 in Tori breed stallions, and 5.5, 7.4 and 5.3, respectively, in Estonian breed stallions. All these testicular measurements were significantly smaller in Estonian than in Tori breed stallions (p < 0.001). Testicular circumference was 45.4 and 35.4 cm in Tori and Estonian breed stallions, respectively (p < 0.001). The testicular circumference was correlated with DSO in both Estonian (p < 0.05) and Tori breed stallions (p = 0.071). The results give us valuable information on the reproductive capacity of Tori and Estonian breed stallions.     

Serum hormones in response to estradiol and (OR) progesterone in ovariectomized cows after thyroidectomy

To evaluate the effect of gonadal steroid treatment and thyroidectomy on concentrations of gonadotropins and thyroid-stimulating hormone in the bovine, nonlactating Holstein cows were either thyroidectomized and ovariectomized (THYOVEX; n=6) or ovariectomized only (OVEX; n=4), and subsequently treated with no gonadal steroids (control), estradiol-17β (E₂), progesterone (P₄), or P₄+E₂ in a 2 × 4 factorial experiment. Averaged across steroid treatments, baseline concentrations of luteinizing hormone (LH; P < .05) and follicle-stimulating hormone (FSH; P <.10) were higher in THYOVEX cows than in OVEX cows. Pulse frequencies and amplitudes of LH and FSH did not differ between THYOVEX and OVEX cows. Secretion of TSH was pulsatile and all concentrations and pulsatile characteristics of TSH were increased (P < .05) in THYOVEX compared to OVEX cows. Treatment with E₂ and P₄ decreased (P < .05) baseline concentrations and magnitude of LH pulses, whereas P₄+E₂ increased (P < .01) pulse frequency of LH and FSH. Amplitude of LH and FSH pulses were not affected by treatment with either steroid. Treatment with P₄+E₂ decreased (P < .05) baseline concentrations of TSH, whereas pulse frequency, and magnitude and amplitude of TSH pulses were not altered by treatment with steroids. Mean concentrations of LH and FSH were similar during 48 hr after termination of E₂ and P₄+E₂ treatments, but concentrations of TSH were higher (P = .06) after P₄+E₂ than after E₂. Secretion of TSH showed a diurnal variation, with the lowest concentrations in the morning and highest in the afternoon. These results indicate that thyroidectomy influenced secretion of gonadotropins in OVEX cows.     

Effects of age,season, and fertility status on plasma and intratesticular immunoreactive (IR) inhibin concentrations in stallions

The nature of the relationship between inhibin and reproductive function in the stallion is yet to be elucidated. Blood and testes from 51 light horse stallions ranging in age from 2 mo to 25 years were collected during the breeding and nonbreeding seasons to study the effects of testicular maturation, aging, season, and fertility status on peripheral and intratesticular concentrations of Ir inhibin and other reproductive hormones. Of the 51 stallions, 12 age-matched stallions (6 fertile, 3 subfertile, and 3 infertile) were used in the fertility study. Blood samples were taken before castration and plasma stored at −20°C for analysis of Ir inhibin, luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T), estradiol (E₂), and estrogen conjugates (EC) by radioimmunoassay (RIA). Testes were homogenized and testicular extracts prepared and frozen at −70°C for analysis of Ir inhibin, T, E₂, and EC by RIA. Plasma concentrations of Ir inhibin, LH, FSH, T, E₂, and EC and intratesticular concentrations of Ir inhibin, T, E₂, and EC increased with age (P < 0.01). The most dramatic effect appeared to be during testicular maturation. An aging effect was not observed in adult stallions. A seasonal effect was not detected for any of the plasma hormones, whereas for the intratesticular hormones the only change noted was an increase in T in the nonbreeding season (P < 0.05). Plasma Ir inhibin, E₂, and EC were lower (P < 0.01) and gonadotropins higher (P < 0.05) in infertile stallions. Plasma T levels did not change. Intratesticular Ir inhibin concentrations tended to be lower (P < 0.1) in subfertile stallions and significantly lower (P < 0.01) in infertile stallions, whereas intratesticular steroid levels were not different among the three groups. In conclusion, plasma and intratesticular Ir inhibin concentrations seem to be affected by testicular maturation and fertility status.     

Plasma concentrations of cortisol, prolactin, luteinizing hormone, and follicle-stimulating hormone in stallions after physical exercise and injection of secretagogue before and after sulpiride treatment in winter

Ten lighthorse stallions were used to determine 1) whether prolactin (PRL) and cortisol responses previously observed after acute exercise in summer would occur in winter when PRL secretion is normally low, 2) whether subsequent treatment with a dopamine receptor antagonist, sulpiride, for 14 d would increase PRL secretion and response to thyrotropin-releasing hormone (TRH) and exercise, and 3) whether secretion of LH, FSH, and cortisol would be affected by sulpiride treatment. On January 11, blood samples were drawn from all stallions before and after a 5-min period of strenuous running. On January 12, blood samples were drawn before and after an i.v. injection of GnRH plus TRH. From January 13 through 26, five stallions were injected s.c. daily with 500 mg of sulpiride; the remaining five stallions received vehicle. The exercise and secretagogue regimens were repeated on January 27 and 28, respectively. Before sulpiride injection, concentrations of both cortisol and PRL increased (P less than .05) 40 to 80% in response to exercise; concentrations of LH and FSH also increased (P less than .05) approximately 5 to 10%. Sulpiride treatment resulted in (P less than .05) a six- to eightfold increase in daily PRL secretion. The PRL response to TRH increased (P less than .05) fourfold in stallions treated with sulpiride but was unchanged in control stallions. Sulpiride treatment did not affect (P greater than .05) the LH or FSH response to exogenous GnRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Acute and Chronic Effects of a Contraceptive Compound RTI‐4587‐073(l) on Testicular Histology and Endocrine Function in Miniature Horse Stallions

The objective of this study was to evaluate acute endocrine effects as well as histological changes in testicular parenchyma induced by the contraceptive compound RTI‐4587‐073(l). Six miniature stallions were used in this experiment. The treatment group (n = 3) received one oral dose of 12.5 mg/kg of RTI‐4587‐073(l), and the control group (n = 3) received placebo only. The stallions' baseline parameters (semen, testicular dimensions, endocrine values) were collected and recorded for 5 weeks before treatment and for 6 weeks after treatment. Multiple blood samples were collected for endocrine analysis. Testicular biopsies were obtained before treatment, 1 day after treatment and every other week after treatment. Ultrasound exams were performed to monitor the dimensions of the stallions' testes. All stallions were castrated 6 weeks after treatment. Sperm numbers, motility and percentage of morphologically normal sperm decreased (p < 0.05), while the number of immature germ cells increased in ejaculates from treated animals (p < 0.05). Serum concentrations of inhibin and follicle‐stimulating hormone did not change. Testosterone concentrations initially transiently decreased (p < 0.05) after administration of RTI‐4587‐073(l), and increased several days later (p < 0.05). Testicular content of testosterone and estradiol 17‐β was lower in treated stallions than in control stallions on Day 1 after treatment (p < 0.05). Severe disorganization of the seminiferous tubules, significant loss of immature germ cells and complete depletion of elongated spermatids were observed in testicular biopsies obtained from treated stallions 1 day, 2 and 4 weeks after treatment. These changes were still present in the testicular samples taken from treated stallions after castration. The results of this study confirmed that RTI‐4587‐073(l) has antispermatogenic effects in stallions. Furthermore, we concluded that this compound causes acute sloughing of immature germ cells from the seminiferous tubules. RTI‐4587‐073(l) has significant but transient effects on Leydig cell function in stallions.     

Effect of administration of adrenocorticotropic hormone on plasma concentrations of testosterone, luteinizing hormone, follicle stimulating hormone and cortisol in stallions

In boars and rabbits, administration of adrenocorticotropic hormone (ACTH) results in a testis-dependent, short-term increase in concentrations of testosterone in peripheral plasma. This experiment was designed to assess the short-term effects of a single ACTH injection on plasma concentrations of testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH) and cortisol in stallions. Eight light horse and two pony stallions were paired by age and weight and then one of each pair was randomly assigned to the treatment (ACTH, .2 IU/kg of body weight) or control (vehicle) group. Injection of ACTH increased (P<.01) plasma concentrations of cortisol by approximately twofold in the first 60 minutes; control stallions showed no change (P>.10) in concentrations of cortisol during the blood sampling period. Control stallions exhibited a midday increase (P>.05) in concentrations of testosterone similar to that reported previously; ACTH treatment prevented or delayed this increase such that concentrations of testosterone in treated stallions were lower (P<.05) than in controls 4 to 5 hours after injection of ACTH. Treatment with ACTH had no effect (P<.10) on plasma concentrations of LH or FSH up to 12 hours after injection.     

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.     

Recommendations for clinical GnRH challenge testing of stallions

Eight mature light-breed stallions with normal testes size, sperm output and semen quality were used to evaluate response to 3 GnRH challenge regimens in the summer in southeast Texas. Gonadotropin releasing hormone (50 μg) was administered intravenously once to each of eight stallions after three days of sexual rest (50 μg GnRH-1X). The same stallions were administered either 5μg GnRH intravenously once hourly for three injections (5 μg GnRH-3X) and 15μg GnRH intravenously once (15μg GnRH-1X) one and two weeks later. Blood samples were collected prior to and at intervals after GnRH administration. Plasma was immediately separated from blood samples and was frozen until assayed for LH, FSH, estradiol and testosterone concentrations. Percentage changes in hormone concentrations from pre-treatment values (baseline) were analyzed by paired studient'st-test to detect significant rises in hormone concentrations. Group mean percentage changes in hormone concentrations were analyzed by analysis of variance to compare responses among treatments. A computerized peak-detection algorithm (PC Pulsar) was used to detect peaks in LH and testosterone concentrations following 5 μg GnRH-3X and 15 μg GnRH-1X treatment.No differences (P>0.10) were detected in percentage change from baseline concentration for LH, FSH, or testosterone at one or two hours after administration of any of the three regimens of GnRH. When more frequent sampling intervals were analyzed for 5 μg GnRH-3X or 15 μg GnRH-1X treatments, no differences were detected in percentage change from baseline concentration for any hormone at 15, 30 or 60 minutes. Thereafter, percentage changes in concentrations of LH and FSH remained increased for 5μg GnRH-3X compared to 15 μg GnRH-1X treated stallions (P<0.05). Percentage changes in concentrations of testosterone were increased for 5μg GnRH-3X compared to 15 μg GnRH-1X treated stallions from 180–300 min (P<0.05), while no differences (P>0.10) were detected between 5 μg GnRH-3X and 15 μg GnRH-1X treated stallions for changes in concentrations of estradiol throughout the experiment.For 15 μg GnRH-1X treated stallions, maximum concentrations of LH in PC Pulsar-detected peaks occurred most commonly at 15 to 30 minutes (7/8 treatment periods) after GnRH injection. Maximum concentrations of testosterone in PC Pulsar-detected peaks occurred most commonly at 60–120 min (7/8 treatment periods) after GnRH injection.A protocol of blood sampling prior to, and 15, 30, 60 and 120 minutes after, intravenous administration of small doses of GnRH would be practical for challenge testing of stallions during the breeding season. In order to reduce cost of hormone assays, we suggest assay of the pre-challenge blood sample (baseline) could include LH, FSH, testosterone and estradiol concentrations (to assess overall hypothalamic-pituitary-testicularfunction), while only LH and testosterone concentrations need be determined after GnRH administration (to assess pituitary and testicular responsiveness). Assay for LH could be done on only the 15 and 30 minute post-GnRH samples, and assay for testosterone could be done on only the 60 and 120 minute post-GnRH samples. Failure to achieve approximately a 50% increase in LH concentration by 30 minutes after GnRH administration, and/or failure to achieve approximately a 100% increase in testosterone concentration by two hours after GnRH administration, could be further pursued either by treatment with increasing dosages of GnRH, or repeated administration of GnRH at hourly intervals, as has been suggested by other workers.     

Effectiveness of controlled internal drug release device treatment to alleviate reproductive seasonality in anestrus lactating or dry Barki and Rahmani ewes during non‐breeding season

This study aimed to evaluate the effectiveness of hormonal treatments on ovarian activity and reproductive performance in Barki and Rahmani ewes during non‐breeding season. Forty‐eight multiparous ewes, 24 Barki and 24 Rahmani ewes were divided into two groups, 12 lactating and 12 dry ewes for each breed. Controlled internal drug release (CIDR) device was inserted in all ewes for 14 days in conjunction with intramuscular 500 IU equine chronic gonadotrophin (eCG) at day of CIDR removal. Data were analysed using PROC MIXED of SAS for repeated measures. Breed, physiological status and days were used as fixed effects and individual ewes as random effects. Barki ewes recorded higher (p < .05) total number of follicles, number of large follicles, serum estradiol concentration and estradiol: progesterone (E₂:P₄) ratio compared to Rahmani ewes. Lactating ewes recorded higher (p < .05) number of small follicles and lower concentration of total antioxidant capacity (TAC) compared to dry ewes. Number and diameter of large follicles recorded the highest (p < .05) values accompanied with disappearance of corpora lutea at day of mating. Serum progesterone concentration recorded lower (p < .05) value at day of mating and the highest (p < .05) value at day 35 after mating. CIDR‐eCG protocol induced 100% oestrous behaviour in both breeds, but Rahmani ewes recorded longer (p < .05) oestrous duration compared to Barki. Conception failure was higher (p < .05) in Barki compared to Rahmani ewes. In conclusion, CIDR‐eCG protocol was more potent in improving ovarian activity in Barki compared to Rahmani ewes, but this protocol seems to induce hormonal imbalance in Barki ewes that resulted in increasing conception failure compared to Rahmani ewes.     

Testosterone propionate treatment of stallions: Effects on secretion of luteinizing hormone and follicle stimulating hormone in daily samples and after administration of gonadotropin releasing hormone

Ten stallions were used to determine if the stallion responds to administration of testosterone propionate (TP) with an increase in follicle stimulating hormone (FSH) secretion after administration of gonadotropin releasing hormone (GnRH) as has been previously observed for geldings and intact and ovariectomized mares. Five stallions were treated with TP (350 μg/kg of body weight) in safflower oil every other day for 11 days; control stallions received injections of safflower oil. The response to GnRH (1.0 μg/kg of body weight) was determined for all stallions before the onset of treatment (GnRH I) and at the end of treatment (GnRH II). Blood samples were also withdrawn daily from 3 days prior to treatment through GnRH II. Treatment with TP decreased (P<.10) concentrations of FSH in daily blood samples. However, treatment with TP did not affect (P>.10) the GnRH-induced secretion of FSH. Concentrations of luteinizing hormone (LH) decreased (P<.05) in daily blood samples averaged over both groups of stallions and were lower (P<.10) in TP-treated stallions than in controls during the latter days of treatment. We conclude that TP administration to stallions does not alter the FSH response to GnRH as has been observed for geldings and for mares of several reproductive states.     

Investigation of in vitro measurable sperm attributes and their influence on electroejaculated bull semen with a fixed‐time artificial insemination protocol in Australian Bos indicus cattle

Increasing use of fixed‐time artificial insemination (FTAI) in beef cattle production has presented an opportunity for the use of fresh or chilled semen as an alternative to standard cryopreserved semen. The objective of this study was to examine in vitro sperm function and pregnancy rate of electroejaculated semen, chilled and stored for 48 hr, compared to conventionally cryopreserved semen with an optimized FTAI protocol in Brahman cattle. Semen from three Brahman bulls was collected, and aliquots were extended in either chilled (at 5°C) or frozen (LN₂) in a Tris‐egg yolk extender base with 2.4% or 7.0% glycerol, respectively. Semen samples were assessed 48 hr after collection or post‐thaw and warming, for sperm motility, in vitro sperm function and fertilizing ability, and used in a FTAI programme. The overall pregnancy rates was significantly different (p < .01) after FTAI with frozen (n = 173; 53.2%) and chilled semen (n = 174; 31.6%). In contrast, the in vitro sperm assessment showed that the chilled semen had significantly faster motility (p < .05), a higher proportion of progressively motile spermatozoa (p < .05), with significantly higher proportions of acrosome intact, viable spermatozoa (p < .01). This study showed that reasonable pregnancy rates in Brahman cattle can be achieved using FTAI with chilled semen collected using electroejaculation and stored for up to 48 hr. However, improvements in semen extenders are required in consideration of semen collection method to improve the longevity of sperm fertilizing ability to significantly increase FTAI output using chilled storage of bull semen.     

Effect of morphine and naloxone on LH response and sexual behavior of rams (Ovis aries)

Effects of the opiate agonist, morphine, and antagonist, naloxone, on LH release, courtship behavior and ejaculation frequency of mature, sexually active or sexually inactive rams were investigated. Plasma LH concentrations were monitored from blood samples collected every 15 min for 10 hr (0800 to 1800 hr) from eight rams that were isolated from or in contact with estrous females. Plasma LH concentration was higher (P<.05) in sexually active rams exposed to receptive females compared with hormone concentration of rams isolated from ewes. Intravenous infusion of morphine sulphate (1 mg/kg) into rams 4 and 6 hr after exposure to ewes reduced (P<.05) plasma LH concentration as compared to rams given saline. Morphine did not affect (P>.05) courtship behavior (investigatory sniff, mount attempt, foreleg kick, flehmen, vocalization) but diminished (P<.05) number of ejaculations. In another trial, LH concentrations were higher (P<.05) in seven sexually active rams given naloxone iv or when given to three rams through an intracerebroventricular cannula (icv) as compared to LH response of sexually inactive rams. LH did not differ (P>.05) in seven sexually inactive rams before or after administration of naloxone. Investigatory sniffs by sexually active rams were increased (P<.03) after treatment with the opiate antagonist. Four of the seven sexually active rams had more ejaculations after naloxone compared with the pretreatment period, but mean ejaculation frequency after treatment did not differ (P=.31). Naloxone did not stimulate courtship behavior of sexually inactive males. These data suggest that the effect of opiates on sexual behavior and LH secretion depends upon the inherent level of sexual activity among rams.     

In vitro studies of Norwegian Red bovine semen immobilized and cryopreserved in alginate solid gel network

Development of new semen cryopreservation techniques improving sperm survival and ensuring availability of viable spermatozoa for a prolonged time‐period after AI is promising tools to reduce sensitivity of timing of AI and enhance overall fertility. The SpermVital^® technology utilizes immobilization of bull spermatozoa in a solid network of alginate gel prior to freezing, which will provide a gradual release of spermatozoa after AI. The objective of this study was to compare post‐thaw sperm quality and in vitro sperm survival over time of Norwegian Red bull semen processed by the SpermVital^® (SV) technology, the first commercialized production line of SpermVital^® (C) and by conventional procedure applying Biladyl^® extender (B). Post‐thaw sperm motility was not significantly different between SV, C and B semen (p > .05). However, sperm viability and acrosome intactness were higher for SV than C and B semen (p < .05). Small differences in DNA quality were observed (p < .05). Sperm viability after storage in uterus ex vivo was higher for SV than for C semen (p < .05). Furthermore, sperm survival in vitro over time at physiological temperature was significantly higher for SV semen than C semen as well as B semen during the incubation period of 48 hr (p < .05). In conclusion, the SpermVital^® technology is improved and is more efficient in conserving post‐thaw sperm quality and results in higher sperm viability over time in vitro for SV than for C and B semen.     

Influences of season and artificial photoperiod on stallions: testicular size, seminal characteristics and sexual behavior

To investigate the influence of daylength on the seasonal reproductive cycle of stallions, 21 stallions were assigned to one of three treatments: control, ambient (natural) photoperiod; S-L, 8 h light and 16 h dark (8:16) for 20 wk beginning July 16, 1982 then 16:8 from December 2, 1982 until March 5, 1984; S-S, 8:16 from July 16, 1982 until March 1984. Temperature was not controlled and was similar for all groups. Total scrotal width (TSW) was measured every 4 wk throughout the experiment. During 10 periods, semen was collected and evaluated every other day for 3 wk and sexual behavior was assessed. The S-L stallions exposed to 16 h light in December had twice as much sperm output in February than in November. Within the February collection period, the sperm output for S-L stallions was greater (P less than .05) than that for either control of S-S stallions. The stimulatory effect of the S-L photoperiod also resulted in larger (P less than .05) testes and decreased (P less than .05) time to ejaculation for S-L stallions in February as compared with either controls or S-S stallions. Despite continued exposure to a 16:8 photoperiod, TSW and sperm output for S-L stallions eventually declined; presumably a consequence of photorefractoriness. The S-S stallions had seasonal cycles coincident with those for control stallions. Based on a sine wave model for TSW and sperm output, stallions in all three groups displayed significant seasonal cycles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of Extent of Apoptosis and DNA Defragmentation in Chilled Semen of Stallions During the Breeding Season

The objective of the study was to assess apoptosis and DNA defragmentation in equine semen diluted and chilled to +4°C. Semen was collected from nine fertile stallions, including four Arabian thoroughbreds and five coldbloods. Examinations were carried out immediately after semen collection (0) and at five storage times (24, 48, 72, 96 and 120 h). The basic semen evaluation was performed in terms of volume, sperm concentration, viable sperm percentage, progressive motility and morphology. Using flow cytometry, DNA defragmentation and cell membrane integrity of spermatozoa were determined. The results of basic tests did not demonstrate significant differences amongst stallions, except for progressive sperm motility, which was significantly higher (p < 0.05) in the semen of Arabian stallions. In the semen of the same stallions, a significant decrease in the percentage of alive spermatozoa was observed at 72, 96 and 120 h of storage, whereas a significant increase in the number of spermatozoa with DNA defragmentation was found after 24 h storage. In the semen of coldblood stallions, significantly reduced live spermatozoa percentage was observed at 96 and 120 h, while increased DNA defragmentation was observed at 48 h. These findings demonstrated that the semen of Arabian stallions chilled to +4°C retained original characteristics until 24 h of storage, whereas in coldbloods, these were preserved up to 48 h of storage.     

Influence of the duration of photoperiod on growth,testicular characteristics and endocrine function of boars

Crossbred boars were used to evaluate the influence of exposure to 8 or 16 hr of light daily from 75 to 175 days of age on growth rate, testicular characteristics and endocrine function. At 160 days of age, concentrations of testosterone in serum (P<.10), the areas under plotted 12 hr testosterone profiles (P<.10) and the number (P<.05) and magnitude (P<.10) of testosterone secretory spikes were increased in boars exposed to 16 hr of light compared to boars in 8 hr light, but concentrations of LH in serum were similar in boars exposed to both treatments. Treatment with GnRH resulted in similar concentrations of LH in serum for both groups of boars. Testosterone in serum after GnRH-mediated LH release was greater at .5 (P<.05) and 1.0 (P<.10) hr following GnRH in boars exposed to 16 hr of light compared to boars at 8 hr, but concentrations of testosterone were similar for both treatments from 1.5 to 4.0 hr after GnRH. Growth rate and testicular and epididymal weights and sperm reserves at 175 days of age were not significantly altered by duration of photoperiod. Boars exposed to 8 hr of light had more hair per unit area than boars exposed to 16 hr of light. We conclude that exposure of prepubertal boars to longer daily photoperiods results in increased concentrations of testosterone in serum at 160 days of age.     

Testicular Volume and Daily Sperm Output in Ragusano Donkeys

A careful evaluation of daily sperm output (DSO) and seminal parameters is indicative of the potential fertility of the animal and at the same time is useful for maximization of the reproductive activity. The aim of this article was to evaluate the seminal parameters, testicular volume (TV) calculated by ultrasound measurements, DSO, and any eventual relationship between these in Ragusano donkey jackasses. Eight Ragusano donkey stallions underwent a morphometric evaluation of the testes by ultrasound to calculate the TV, and daily semen collection for 10 consecutive days. TV ranged from 250 to 500 cm³ and no significant differences could be observed between the left and right testes. Only ejaculates from 4 to 10 days were analyzed, given that there was evidence that the sperm extragonadal reserve interfered with the total number of sperm in ejaculates during the early days. DSO in the Ragusano donkey ranged from 7 to 23 × 10⁹ sperms. The seminal parameters showed high values of motility, viability, and low abnormality percentages. Age, TVs, and sperm parameters were not significantly correlated. The correlation between TV and DSO was poor (P = .1) in this study and this may suggest that TV and DSO might simply not be highly correlated in the donkey.     

Trypan Blue/Giemsa Staining to Assess Sperm Membrane Integrity in Salernitano Stallions and its Relationship to Pregnancy Rates

Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty‐one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty‐five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p < 0.05) and visual motility (p < 0.01). A decrease in sperm motility, concentration (p < 0.05) and total sperm number was found in June–July (p < 0.01). Live sperm with intact acrosome (LSIA) and proximal droplets (PD) were lower (p < 0.01) in June–July, while acrosome reacted sperm (ARS) percentage increased (p < 0.05). No fertility differences were found among stallions with an average fertility per cycle of 44.6% and a pregnancy rate of 68.6%. Higher percentages of LSIA were found in the ejaculates used to inseminate mares that became pregnant vs those used in mares not pregnant (p < 0.05). The significance of LSIA as test variable to verify the reliability of Trypan blue/Giemsa staining was confirmed by Receiver operating characteristic ROC analysis and the sensitivity of the test was 85% at a cut‐off value of 48% LSIA. Trypan blue‐Giemsa showed to be an accurate method that can be applied on field to evaluate sperm membrane integrity and to identify poor‐quality ejaculates.     

Purification of cryopreserved camel spermatozoa following protease-based semen liquefaction by lectin-functionalized DNA-defrag magnetic nanoparticles

Although incorporating proteases into sperm medium is considered the most effective procedure to eliminate camel semen viscosity, it drastically affects viability, morpho-functional properties and, hence, fertilization potential of spermatozoa. The present work aimed at evaluating adequacy of employing magnetic nanoparticles-based sperm purification technique for eluting impaired and apoptotic camel spermatozoa from cryopreserved semen doses following protease-based semen liquefaction. Thirty cryopreserved semen doses (50 x 10⁶ sperm/straw) representing the following liquefaction treatments: control (untreated), 0.1 mg/ml papain or 5 U/ml bromelain were used (n = 10 straws per treatment). Immediately after thawing (38°C for 40 s), sperm concentration of each straw within treatment was readjusted to 15 x 10⁶ sperm/mL by dilution in PBS (37°C). Sperm physical and cytological properties were then assessed (non-purified semen). Thereafter, each specimen was subjected to lectin-functionalized DNA-defrag magnetic nanoparticles sperm purification, and the same sperm traits were re-evaluated after undergoing purification (purified semen). Sperm DNA fragmentation level within each group, prior to and after magnetic nano-purification, was also determined by fluorescent imaging. The results showed a dramatic improvement (p < .05) in post-thaw motility (%), viability (%), normal sperm (%), intact acrosome (%) and HOST-reacted (%) spermatozoa in protease-liquefied semen following sperm magnetic nano-purification. Additionally, the highest (p < .05) DNA fragmentation level was recorded in all cryopreserved semen groups prior to purification, whereas the lowest (p < .05) was observed in the protease-treated specimens after magnetic nano-purification. These results indicate that protease-based semen liquefaction prior to cryopreservation in conjunction with magnetic nano-purification post-thawing holds potential for reducing the proportion of damaged and dead spermatozoa, hence improving camel sperm fertilization competence.