Resuspending ram spermatozoa in seminal plasma after cryopreservation does not improve pregnancy rate in cervically inseminated ewes

The role of seminal plasma (SP) components on the maintenance of motility, viability and fertilising ability of frozen-thawed spermatozoa is of considerable interest. However, differences observed in constituents of SP among males could explain differences in fertility obtained in vivo. Two experiments were designed to examine the effects of seminal plasma on fertility from cervically inseminated frozen-thawed semen. The objective of Experiment 1 was to investigate if source or type of SP influences pregnancy rate. Seminal plasma was collected from rams previously classified as having either High (HSP; n=3) or Low (LSP; n=3) fertility in vivo. Artificial SP (fructose/sodium solution with 10% BSA; ASP) was made. Frozen semen from the same 6 rams was thawed and inseminated (Control) or resuspended either in HSP, LSP or ASP (20% in semen) prior to insemination of ewes (n=284, over 2 farms). The overall pregnancy rate was 28.1%. Treatments (Control, ASP, HSP and LSP) were not significantly different (P>0.3). There was no difference between HSP and LSP (P>0.5), and no effect of using ASP compared to ram SP (P>0.7), on pregnancy rate. As there was no effect of SP on pregnancy rate a repeat experiment (Experiment 2) was designed to test the effect of washing and selecting motile sperm prior to resuspending in phosphate-buffered saline (PBS) containing SP on pregnancy rate. Frozen-thawed semen from each of 2 rams was centrifuged through a density gradient, pellets were centrifuged through a wash medium and the sperm concentration/ram was counted. Sperm cells were resuspended in: (1) control PBS, (2) PBS containing 30% HSP or (3) PBS containing 30% LSP to give 100 x 10(6) motile sperm in 0.25 mL. Control straws were thawed and inseminated directly. Ewes (n=223 over 2 farms) were inseminated 57 h post-sponge withdrawal and those not returning to oestrus were slaughtered 29-50 days post-insemination for pregnancy determination. In Experiment 2, the pregnancy rate for Control, PBS, HSP and LSP were 15.4%, 2.3%, 0% and 0%, respectively, for Farm 1 (P>0.05) and 17.8%, 11.0%, 3.9% and 12.4%, respectively, for Farm 2. Under the conditions of the current study, addition of SP from different donors of either High or Low fertility status to frozen-thawed ram semen post-thawing did not improve pregnancy rate in ewes. ASP had no effect on pregnancy rate in ewes when added to frozen-thawed semen. Washing and selection of motile sperm prior to resuspension in PBS with or without SP (30%) before insemination had a negative effect on pregnancy rate in cervically inseminated ewes. Hence, the addition of seminal plasma or some of its constituents to semen does not appear to improve pregnancy rate in cervically inseminated ewes.     

Ram seminal plasma improves pregnancy rates in ewes cervically inseminated with ram semen stored at 5 °C for 24 hours

In this study, we compared pregnancy rates obtained using ram semen stored at 5 °C for 24 h, with ram or bull seminal plasma (SP) added to TRIS-egg yolk extender. During the breeding period, 670 adult Corriedale ewes were cervically inseminated with semen (2 × 10⁸ sperm in a volume of 0.2 mL) from eight adult Corriedale rams. Ejaculates, obtained using an artificial vagina, were split into three aliquots and diluted with the following: TRIS-egg yolk based extender (T), T + 30% ram SP (R), or T + 30% bull SP (B). Samples were refrigerated and stored at 5 °C for 24 h until used for AI. Pregnancy was assessed by ultrasonography 35 to 40 d after AI. Pregnancy rate was not affected by ram (P = 0.77) or breeding period (P = 0.43), and there were no interactions between extender and ram (P = 0.94), or extender and breeding period (P = 0.24). However, there was an effect of extender (P = 0.0009) on pregnancy rates; ram SP, but not bull SP, increased pregnancy rates compared with extender without SP (49.7, 38.1, and 31.1%, for R, B, and T respectively). In conclusion, ram SP added to TRIS-egg yolk extender had a beneficial effect on the pregnancy rate of ram sperm stored at 5 °C for 24 h and used for cervical insemination of ewes.     

Conserved ram seminal plasma proteins bind to the sperm membrane and repair cryopreservation damage

Whole seminal plasma (SP) enhances the function and fertility of frozen/thawed ram sperm. The objective of the current study was to investigate whether SP proteins capable of binding to molecules from the sperm plasma membrane were conserved among ram breeds, and whether these proteins were sufficient to overcome cryopreservation-induced reductions in sperm quality. Whole ram SP, obtained from rams of various breeds, improved progressive motility of frozen/thawed sperm at all times evaluated (P < 0.05); however, it did not improve total motility (15 min, P = 0.480; 30 min, P = 0.764; and 45 min, P = 0.795). To identify SP proteins responsible for this effect, a new method was developed to retain SP proteins that bound specifically to the sperm membrane by immobilization of sperm membrane proteins. These proteins specifically bound to the sperm surface, especially the acrosomal region. Lactotransferrin, epididymal secretory protein E1, Synaptosomal-associated protein 29, and RSVP-20 were identified (mass spectrometry) in this fraction. The retained SP proteins fraction repaired ultrastructural damage of frozen/thawed sperm and, with the addition of fructose, significantly improved motility of frozen/thawed sperm. We concluded that SP proteins that bound to the sperm membrane were conserved among ram breeds, and that when added to frozen/thawed semen (along with an energy source), they repaired ram sperm damage and enhanced sperm motility.     

Morphometrically-distinct sperm subpopulations defined by a multistep statistical procedure in ram ejaculates: intra- and interindividual variation

The existence of sperm subpopulations within the mammalian ejaculate has now been widely recognized. However, to the best of our knowledge, no data exist regarding the existence of sperm morphometric subpopulations within the ovine ejaculate. Computer assisted sperm morphometry analysis (ASMA) data and clustering methods were used in this study to identify sperm-head subpopulations in ram semen. Two experiments were carried out. In Experiment 1, ejaculates from 226 mature rams of the Manchega breed belonging to 36 different herds were used. A minimum of 100 sperm heads were analyzed from each male and eight morphometric characteristics for each individual sperm were recorded. Subpopulation analysis was performed in sequential steps: variable group analysis and correlation analysis to select which morphometric characteristics to use in cluster analyses; nonhierarchical clustering analysis using sperm head length and p2a (also known as roundness) shape factor as initial classificatory variables; and hierarchical clustering analysis to obtain the final number of clusters. The clustering analyses, based on 26 306 individual cells, revealed the existence of four sperm subpopulations (SP1, SP2, SP3 and SP4) with different morphometric characteristics. Significant differences in the proportion of spermatozoa in the SP1 and SP3 were found between rams belonging to different herds. In Experiment 2, the intra- and intermale variability on the distribution of sperm subpopulations was assessed. Three ejaculates from each of 21 rams were collected and the same multistep clustering analysis was performed. For all subpopulations defined, the intermale variability resulted in high values, being the intramale variability much lower. This fact would allow the use of sperm head morphometry to characterize a male and might provide valuable information to asses its fertility. In conclusion, our results show that using computer assisted sperm morphometry analysis and multivariate cluster analyses, four sperm subpopulations with different head phenotype were identified in ram ejaculates.     

Relationship between in vitro sperm functional tests and in vivo fertility of rams following cervical artificial insemination of ewes with frozen-thawed semen

Several procedures have been proposed to assess structural and functional characteristics of cryopreserved ram semen but none so far have yielded consistent relationships with in vivo fertility. The objectives of this study were to evaluate several sperm function tests as potential markers of in vivo ram fertility (determined by pregnancy rate in ewes) using frozen-thawed semen. In experiment 1, frozen-thawed straws (n=3 per ram) of semen from three high and three low fertility rams were assessed using fluorescent microscopy for (1) progressive motility, (2) viability and, (3) acrosomal status. In experiment 2, frozen-thawed straws (n=3 per ram) of semen from 18 rams of known fertility were analysed using either computer-assisted sperm analysis (CASA) for eight motion characteristics or flow cytometric staining for: (1) viability and acrosomal status, (2) plasma membrane status and capacitation-like changes, and (3) live cells following an osmotic resistance test (ORT). In experiment 3, platelet-activating factor (PAF) was isolated from straws (n=2 per ram) of semen using high-pressure liquid chromatography (HPLC) and quantified using HPLC-tandem mass spectrometry for 18 rams. In experiment 1, no association was found between motility, viability (% live) or acrosomal status (% damaged, % intact and % reacted) and in vivo fertility. In experiment 2, no correlation was found between motility (CASA), viability (% live), acrosomal status (% live, % live intact and % reacted), capacitation status (% capacitated, % non-capacitated), plasma membrane stability (% dead) and % live cells following ORT and ram in vivo fertility. In experiment 3, there was no relationship between PAF content in spermatozoa and ram fertility. In conclusion, we were unable to relate the in vivo fertility of rams with in vitro functional tests of their frozen-thawed semen and suggest that the fertility of a given semen sample cannot easily be quantified using available in vitro tests.     

Freezability of spermatozoa from Finn and Dorset rams in multiple semen extenders

Ten semen extenders were tested in two experiments for cryopreservation of semen collected from four Finn and four Dorset rams. Two ejaculates of semen were combined from each ram for testing each extender treatment. The extenders consisted of a series of commonly used egg yolk-TRIS media with and without sodium and triethanolamine lauryl sulfate (STLS), a similar extender with 3-N-morpholino propane sulfonic acid (MOPS), and milk and whey extenders. In Experiment 1, extender treatments were replicated with three sets of collections from the eight rams, and in Experiment 2 with two sets. The egg yolk-TRIS-glycerol-STLS (EY(1)TSTLS) extender was significantly superior to other extenders except whole milk in protecting the sperm during freezing and thawing. In Experiment 1, a 20% egg yolk-TRIS-glycerol-STLS extender preserved 71% of the progressively motile Finn sperm (post-thaw divided by pre-freeze percentage of motile sperm), and 76% of the Dorset sperm. In Experiment 2, the corresponding values for the same EY(1)TSTLS extender used with Finn and Dorset sperm were 86 and 64%, respectively. Without STLS the egg yolk extenders were significantly less effective in protecting cryopreserved ram sperm. This egg yolk-TRIS extender, containing STLS and glycerol, may hold promise for freezing ram sperm that could be used successfully for intracervical insemination.     

Short term, repeated exposure to rams during the transition into the breeding season improves the synchrony of mating in the breeding season

The ram effect is widely used in Mediterranean breeds of sheep but its use in temperate genotypes is restricted by breed seasonality. However, ewes from these highly seasonal genotypes are sensitive to stimulation by rams close to the onset of the natural breeding season. In this study we developed a pre-mating protocol of repeated, short-term exposure to rams (fence-line contact or vasectomised rams) beginning during late anoestrus and continuing into the breeding season. We hypothesised that this pre-mating protocol would synchronise the distribution of mating of North of England Mule ewes during the breeding season above that observed in ewes isolated from rams prior to mating. Ram-exposed ewes were given contact with rams (Experiment 1: fence-line; FR, n = 94 and Experiment 2: vasectomised rams; VR; n = 103) for 24 h on Days 0 (10 September), 17 and 34 of the experiment. Control ewes (Experiment 1; FC, n = 98 and Experiment 2; VC; n = 106) remained isolated from rams prior to mating. In Experiment 2, a subset of VR (n = 35) and VC ewes (n = 35) were blood sampled twice weekly to monitor their pre-mating progesterone profiles. At mating, harnessed entire rams were introduced, 17 or 16 days after the last ram exposure (Experiments 1 and 2) and raddle marks were recorded daily. The median time from ram introduction to mating was reduced in ewes given both fence-line and vasectomised ram contact (P < 0.001), leading to a more compact distribution of mating and lambing (At least P < 0.01). In the blood sampled VR ewes, there was a progressive decline in the number of days from ram exposure to the onset of dioestrus (at least P < 0.05). This observation indicates that the cycles in VR ewes became increasingly synchronised over the pre-mating period, a pattern not evident in VC ewes. In conclusion, repeated, short-term exposure of ewes to rams during the transition into the breeding season is an effective method of synchronising the distribution of mating during the breeding season.     

Relationships between Brucella ovis semen culture and various semen and serology parameters

Semen and blood samples from 154 rams from two Montana range flocks (Flock A, vaccinated for Brucella ovis ; Flock B, nonvaccinated) were evaluated to determine the relationship between Brucella ovis (B. ovis ) semen culture results and various semen and blood parameters. All rams utilized in this study exhibited no palpable ram epididymitis lesions. Thirteen and 25.6% of the rams tested in Flocks A and B, respectively, had positive B. ovis semen cultures. Only age of ram and ram condition scores differed (P<0.05) between flocks. No flock by semen culture interactions were detected (P>0.05) for any of the parameters evaluated. Age of ram, ram condition score, and spermatozoa rate from forward movement were unrelated (P>0.05) to B. ovis culture results. Rams with positive B. ovis semen cultures had lower sperm motility (P<0.05), higher percentage of abnormal spermatozoa cells (P<0.05), higher percentage of spermatozoa head abnormalities (P<0.01), lower percentage of live-normal cells (P<0.05), higher incidence of white blood cells in semen (P<0.01) and higher complement fixation (CF) titers (P<0.01).     

Assessment of post-thawed ram sperm viability after incubation with seminal plasma

A suggested alternative to improve post-thawed ram semen quality is the addition of seminal plasma (SP). This is thought to be capable of improving sperm resistance to thermal shock, reverting cryocapacitation and helping sperm survival. The aim of this study was to evaluate the effect of frozen-thawed ram semen incubation with SP on mitochondrial activity, acrosomal membrane integrity, necrosis and apoptosis. Frozen/thawed semen was divided into two groups: the SP Group and the control group. After 0, 30 and 60 min, fluorescent probes were added to aliquots from each treatment group and evaluated using flow cytometry. There was no difference between treatment groups in almost all viability parameters evaluated, with exception of the apoptosis, which was found increased in SP group. The increase in incubation period resulted in a decreased percentage of sperm with high mitochondrial membrane potential and acrosomal membrane integrity and an increased percentage of necrotic and apoptotic sperm cells. In conclusion, the present study showed that addition of seminal plasma after thawing cryopreserved ram sperm had no identifiable beneficial effect on sperm quality.     

An acrosin inhibitor in ram spermatozoa that does not originate from the seminal plasma.

Ram seminal plasma, and ejaculated ram spermatozoa that have been washed with 0.25M sucrose, both contain acrosin inhibitor. The aim of this work was to determine whether the intracellular inhibitor originates from the seminal plasma. The amounts of inhibitor in ejaculated and epididymal spermatozoa were measured and compared with the amounts present in the seminal plasma of normal and vasectomized rams. One ejaculated ram spermatozoon contained 2.1 amol (2.1 X 10(-18) mol) of inhibitor and one epididymal spermatozoon contained 3.3 amol of inhibitor. (All molarities are mean values based on pooled ram semen or on single ejaculates from three vasectomized rams.) Calculations from results in earlier publications indicated that one ejaculated ram spermatozoon contains about 3 amol of acrosin; thus the inhibitor: acrosin ratio in washed ram spermatozoa is approximately 1. One ml of ram semen contains, on average, 3 X 10(9) spermatozoa and not more than 0.8 ml of seminal plasma. This number of ejaculated spermatozoa would contain 6.3 nmol of inhibitor, while the same number of epididymal spermatozoa would contain 9.9 nmol of inhibitor. These values exceed the quantities of inhibitor present in 0.8 ml of normal seminal plasma (approximately 1.6 nmol) or in 0.8 ml of seminal plasma from vasectomized rams (approximately 2.3 nmol). We conclude that seminal plasma is not a major source of the acrosin inhibitor that can be recovered from washed ejaculated ram spermatozoa.     

Assessment of ram sperm membrane integrity following different thawing procedures

Semen from five 2.5-yr-old rams selected for use in an AI program was collected over 3 consecutive days using an artificial vagina. The semen was diluted with a skim milk extender containing 7% glycerol (v/v), packed in French mini-straws (approx. 100 mill/straw), and frozen in a programmable freezer. Three freezing operations were carried out per ram. Three straws per freezing operation were subjected to the following thawing procedures: 1) 70 degrees C, 5 sec; 2) 50 degrees C, 9 sec and 3) 35 degrees C, 12 sec. Post-thaw sperm motility was subjectively assessed using a phase contrast microscope; while the combined fluorochromes carboxyfluorescein diacetate and propidium iodide (CFDA/PI), the hypo-osmotic swelling test (HOS) and the presence of normal apical ridges (NAR's) were used to determine the degree of sperm membrane integrity. Significant differences between thawing treatments were found for post-thaw motility (P < .05) and membrane integrity (P < 0.01), and variation among rams was statistically significant. Post-thaw sperm motility as well as the percentage of spermatozoa showing intact membranes were significantly higher (P < 0.01) for straws thawed at 70 degrees C than for those thawed at 35 degrees C (67.0 +/- 1.1 and 63.0 +/- 1.1%, and 50.5 +/- 1.5 and 41.7 +/- 1.5%, respectively). However, no corresponding statistically significant difference could be found for these parameters when 70 degrees C and 50 degrees C thawing were compared. It was concluded that sperm can be thawed at 50 degrees C for 9 sec instead of 70 degrees C for 5 sec without further reducing sperm motility or membrane integrity. This lower thawing temperature would facilitate the widespread use of frozen/thawed ram semen under farm conditions in Sweden.     

Seasonal variations in the composition of ram seminal plasma and its effect on frozen-thawed ram sperm

It has been proposed that seminal plasma (SP) in the extender or in post-thaw media can prevent and revert cold-shock damage in cryopreserved ram sperm; however, this was dependent on season. We evaluated sperm parameters from Frisian ram semen incubated for various intervals with SP from all seasons and stored at -18 or -196 degrees C. At both temperatures, SP from autumn or winter increased (P<0.05) sperm motility, whereas no SP, or SP from spring or summer, had no effect. However, neither viability nor membrane or acrosomal status were modified by SP. Thirteen SP proteins were bound to the sperm surface (16.1, 16.7, 17.4, 23.3, 25.2, 27.5, 35.0, 40.0, 49.0, 53.5, 55.5, 61.0, and 86.0kDa). The SP proteins that bound to sperm were affected by season, but not by conservation temperature. Sperm incubated with SP from autumn had increased concentrations of five proteins; two were identified (with specific antibodies) as RSVP14 and RSVP20. In conclusion, SP from autumn and winter improved sperm motility of frozen-thawed ram sperm, and storage of ram SP at -18 or -196 degrees C did not affect protein composition. The SP proteins that bound to the sperm surface may be responsible for sperm membrane stabilization and should be further investigated.     

Ram novelty and the duration of ram exposure affects the distribution of mating in ewes exposed to rams during the transition into the breeding season

This study compared the affect of short-term and continuous exposure to rams during the transition between anoestrus and the breeding season on the distribution of mating and subsequent lambing. Further, within ewes continuously exposed to rams we investigated the effect of replacing these rams every 17 days with 'novel' rams. During August (late anoestrus, Northern Hemisphere), multiparous, North of England mule ewes were allocated to one of four groups: SVR ewes were exposed to vasectomised rams for 24h on Day 0 (short term; n=109), RVR ewes were exposed to vasectomised rams for 24h on Days 0, 17 and 34 (short term; n=113); PVR ewes were exposed to vasectomised rams on Day 0 and remained with the same rams for the duration of the pre-mating period (continuous; n=104); NVR ewes were continuously exposed to vasectomised rams from Day 0 with the rams replaced with 'novel' rams every 17 days (continuous; n=113). Blood samples were collected from a subset of ewes (n=22 per group) to monitor progesterone. On Day 50, harnessed, entire rams were introduced for mating and raddle marks recorded daily for the first 17 days. The median date of mating occurred 1 day earlier in NVR ewes than PVR ewes (P<0.05). A synchrony score calculated from the blood sampled ewes showed that the distribution of mating was more synchronised in PVR and NVR ewes than SVR and RVR ewes (P<0.001). PVR and NVR ewes had an earlier onset of cyclic activity than RVR ewes (P<0.01). However, only NVR ewes differed from SVR ewes in this variable (P<0.05). Within ewes lambing to first service, the median date of lambing of PVR, NVR and SVR ewes occurred at least 2 days earlier than RVR ewes (at least P<0.05). Further, PVR and NVR ewes had a more compact distribution of lambing than SVR and RVR ewes (P<0.05) and lambing was more compact in NVR ewes than PVR ewes (P<0.05). In conclusion, ewes in continuous contact with rams prior to mating had a more synchronised distribution of mating and lambing than ewes given only short-term exposure to rams. This distribution of mating in continuous ram exposed ewes can be further enhanced by periodic exposure to novel rams.     

Concentration of prostaglandins E and F,fructose and glycerylphosphorylcholine in ram semen obtained by electro-ejaculation or artificial vagina and in vesicular fluid

The concentration of spermatozoa in electrically ejaculated ram semen was lower than in semen obtained by an artificial vagina. Glycerylphosphorylcholine concentrations were also lower in the electrically ejaculated semen and there was a high correlation between sperm and glycerylphosphorylcholine concentrations.Seminal fructose, prostaglandin E (PGE) and prostaglandin F (PGF) levels did not differ significantly between the two methods of collection but there was greater variability between rams when they were electrically ejaculated.The concentration of fructose in the vesicular secretion of rams was less variable and higher than in seminal plasma whereas PGE or PGF concentrations were not significantly different in the two fluids.     

Liquid storage of flow cytometrically sorted ram spermatozoa

This study investigated the optimum short-term storage conditions for ram spermatozoa before and after flow cytometric sorting. Prior to sorting, semen from four rams (n = 3 ejaculates per ram) was diluted in either a Tris-based diluent (TRIS) or AndroHep (AH) and stored at 5, 15 or 21 degrees C for 0, 6 or 24h. Sperm characteristics were assessed during storage and after sorting, freeze-thawing and incubation (6h, 37 degrees C). Functional capacity and migration ability in artificial cervical mucus (sperm migration test (SMT)) of stored, sorted and non-sorted (control) spermatozoa were assessed after freeze-thawing. After sorting, semen from three rams (n = 3 ejaculates per ram) was diluted in four different extenders: ultra-heat-treated (UHT) long life milk, TRIS containing 10% (v/v) egg yolk (TRIS-EY), AH (pH 7.4), or TEST buffer containing 10% (v/v) egg yolk (TYB). Sorted and non-sorted (control) spermatozoa were stored at 15 degrees C for 24h or 5 degrees C for 6 days. Sperm characteristics were evaluated at 0, 6 and 24h for samples stored at 15 degrees C and daily for samples stored at 5 degrees C. The SMT was performed on sorted and non-sorted (control) spermatozoa after 6h and 3 days storage at 15 and 5 degrees C, respectively. Spermatozoa stored in TRIS were sorted more efficiently, had higher motility after sorting, freezing, thawing and incubation and had greater numbers of spermatozoa penetrating into the SMT than spermatozoa stored in AH prior to sorting. Spermatozoa stored in UHT at both temperatures had higher motility, acrosome integrity and traveled greater distances in the SMT than spermatozoa stored in all other diluents. In summary, storage in TRIS at 21 degrees C was optimal for transport of ram spermatozoa to the sorting site, and storage of spermatozoa in UHT diluent (after sorting) preserved sperm viability and migration ability best at both 15 and 5 degrees C.     

Semen characteristics of Ile-de-France rams of different age and physical condition

A breeding soundness examination (BSE) involving animal physical examination, scrotal circumference (SC) and semen evaluation was undertaken on 80 Ile-de-France rams at a government breeding farm, 32 km south-west of Casablanca (Morocco) from March to May 1988. A large percentage of rams (21.4%) was found to be unfit for breeding due to physical and genital abnormalities; 11 and 5% had disorders of the feet and respiratory system; upon genital palpation, 17.5, 13.8 and 7.5% of animals had orchitis, epididymitis and posthitis, respectively. The SC increased with age from 28.8+/-3.2 cm at 相似文献   

Effects of levamisole on hyaluronidase activity and sperm characteristics in rams

The aim of this study was to determine the effects of levamisole on sperm characteristics and hyaluronidase activity of blood serum and semen. For this purpose, 12 Akkaraman rams (2-3 years old) were used. Levamisole hydrochloride was administered orally at a dose of 7.5mg/kg body weights once daily for 2 days. Serum and semen samples were collected from the rams at post-treatment 1, 2, 4, 24, 48, 72, 96, 120, 144, 216, 288 and 384 h and examined for sperm characteristics and hyaluronidase activity. The results showed that the use of levamisole caused significant (P < 0.01) increase in serum hyaluronidase activity at all times except the 72 h, and in semen hyaluronidase activity at 1, 2, 4, 24, 72, 96 and 120 h compared to the control group. In addition, the levamisole caused significant (P < 0.05) decreases in semen volume, sperm motility, concentration and total sperm number at all times. There was no correlation between semen hyaluronidase activity and the sperm characteristics. In conclusion, levamisole did not have any deleterious effect on hyaluronidase enzyme. However, the use of this drug in rams during the breeding season is harmful due to the decrease of sperm characteristics.     

The effect of a zinc, cobalt and selenium soluble glass bolus on trace element status and semen quality of ram lambs

Supplemental zinc and selenium were administered to ram lambs grazed on pastures that were not considered to be deficient in either element. The breeding season and polygamy of the ram mean that his requirements for semen production will be relatively large over a short breeding season and this may induce a localised deficiency of zinc and/or selenium, thus resulting in a decrease in semen quality and production.Thirty-three 8-month-old ram lambs were kept at grass and fed a supplement of barley and peas, with ad libitum access to grass silage when grazing became restricted. On day 0, the rams were allocated to two groups by restricted randomisation of live weight. One group each had a zinc, cobalt and selenium soluble glass bolus (Zincosel(R), Telsol) administered with the other group not receiving a bolus to act as a control. Blood samples were taken by jugular venipuncture at day 0 (prior to bolus administration) and at days 23, 44, 65 and 86. Blood samples were analysed for zinc status (plasma zinc concentration) and selenium status (erythrocyte glutathione peroxidase activity). Semen was collected once a week between days 44 and 86, by diversion during a natural mount. Semen quality was assessed by ejaculate volume, spermatocrit, sperm concentration, abnormal morphology, motility, percentage live (negrosin-eosin stain), membrane integrity (hypo-osmotic swelling test (HOS)) and seminal fluid glutathione peroxidase activity and zinc concentration. The bolused lambs had a significantly increased erythrocyte glutathione peroxidase activity (P<0.01) on all samplings after bolusing and had significant increases in motility, proportion of live sperm and proportion of intact membranes indicated by the HOS. The bolused ram lambs had an increased selenium status and apparent improvement in semen membrane quality.     

Relationship between in vitro fertilisation of ewe oocytes and the fertility of ewes following cervical artificial insemination with frozen-thawed ram semen

No laboratory test exists that can reliably predict differences among rams in field fertility after artificial insemination (AI) with frozen-thawed semen. In vitro fertilisation (IVF) has been proposed as a method of predicting these differences. The objectives of this study were to evaluate whether IVF system could discriminate among rams of different fertility in vivo after AI using frozen-thawed semen. Also, to examine effects of lowering sperm concentration on discrimination power between rams used for IVF. The aim of Experiment 1 was to evaluate the effect of altering the sperm concentration from 2 x 10(6) to 0.03125 x 10(6) spermatozoa/mL on subsequent cleavage rate and blastocyst rate in vitro. In Experiment 2, six rams (three High and three Low in vivo fertility; average pregnancy rates of 37.6% and 21.8%, respectively) were compared for their fertilising ability in IVF. Spermatozoa from each of the six rams were added to ewe oocytes using a concentration of either 2 x 10(6) or 0.0625 x 10(6)/mL. There were six replicates with 25 oocytes per well and two wells per ram per replicate. Cleavage rate was monitored at 48 h post-insemination (p.i.) and blastocyst rate determined on Days 6-8 p.i. In Experiment 1, cleavage rate increased with increasing sperm concentration and blastocyst rate was not affected by sperm concentration on any day. When the six rams were tested using 2 x 10(6) spermatozoa/mL, no significant differences were found between High and Low fertility groups for cleavage rate or blastocyst rate on Days 6, 7, or 8 p.i. (P>0.05). When the experiment was repeated using 0.0625 x 10(6) spermatozoa/mL, no differences were found between High and Low group rams for blastocyst rate on any of Days 6, 7 or 8 p.i. (P>0.05). However, there was a significant difference between High and Low fertility rams for percentage of oocytes cleaved (16.4, S.E. 2.02%; P<0.01) and the correlation between fertility in vivo and cleavage rate in vitro was significant (P=0.013). Replicate of IVF was a source of significant variation for both cleavage rate and blastocyst rate and conditions need to be further controlled. However, we suggest that using a low concentration of spermatozoa (0.0625 x 10(6)/mL) for IVF may be a useful method for predicting field fertility of frozen-thawed ram semen.     

The effect of dietary n-3 polyunsaturated fatty acids supplementation of rams on semen quality and subsequent quality of liquid stored semen

The objective of this study was to examine the effect of dietary n-3 polyunsaturated fatty acid (PUFA) supplementation of rams on semen quality and subsequent sperm function of liquid stored semen. Mature rams of proven fertility were individually housed and were blocked according to breed, body weight, and body condition score and randomly allocated within block to one of two dietary treatments (N = 7 per treatment). Rams were offered a base diet of hay and concentrate, with the concentrate enriched with either: (1) saturated palmitic acid (CON) or (2) high n-3 PUFA fish oil (FO) supplements. Both lipid supplements were added at 2% (wt/wt) of the total diet as fed and both were partially rumen-protected. The animals were fed their respective diets for a total of 9 weeks and blood samples were collected on weeks 0 (pre-experimental), 4, and 9, relative to initial allocation of diet (week 0), for measurement of plasma concentration of fatty acids, metabolites, insulin like growth factor 1 (IGF-1) and insulin. Semen was collected from each ram (on 1 day in each week) in weeks 4, 5, 7, 8, and 9, and each ejaculate was assessed for volume, wave motion, and concentration of sperm, after which it was diluted in a skim milk-based extender and stored at 4 °C. A second ejaculate was collected on weeks 4, 7, and 9, centrifuged, and the sperm frozen for subsequent lipid analysis. A sample of semen from each ram was assessed at 24, 48, and 72 hours after collection for sperm progressive linear motion, ability to penetrate artificial mucus, and the ability to resist lipid peroxidation (at 24 and 48 hours only) using the thiobarbituric acid reactive substances assay. There was no effect of diet on plasma insulin concentrations or on any of the metabolites measured, however, there was a diet by week interaction for plasma IGF-1 concentration (P < 0.05). This was manifested as the FO supplemented rams having higher IGF-1 concentrations on week 9 compared with the control treatment (P < 0.05), but not at the earlier sampling dates. Compared with the pre-experimental values, supplementation with FO increased plasma concentrations of total n-3 PUFAs by 3.1-fold and decreased n-6 PUFA concentrations by 1.84-fold. Consequently, the ratio of n-6 to n-3 PUFA was decreased in the FO-supplemented rams (P < 0.001). Dietary supplementation with FO increased the concentration of eicosapentaenoic acid in sperm from week 4 to 9 by 2.7-fold (P < 0.05) leading to a 1.5-fold increase in total n-3 PUFA in the same period. Ejaculates collected from rams supplemented with FO yielded a higher semen concentration (P < 0.05), however, there was no difference between diets on any of the other semen quality parameters including semen volume, wave motion, progressive linear motion, ability to penetrate artificial mucus, or ability to resist lipid peroxidation. In conclusion, dietary supplementation of rams with n-3 PUFA successfully increased the n-3 PUFA content of plasma and sperm but has limited effects on the quality of liquid stored semen.