A.I. Peña

Effects of Equex from different sources on post-thaw survival, longevity and intracellular Ca2+ concentration of dog spermatozoa

The aims of the present study were to compare the effects of two commercial preparations (Equex STM Paste or Equex Pasta), whose active ingredient is sodium dodecyl sulphate (SDS), added to a Tris-egg yolk-based extender, on post-thaw sperm survival and longevity, as well as on the intracellular Ca(2+) concentration of dog spermatozoa during incubation at 38 degrees C. One ejaculate was collected from each of eight dogs. Each ejaculate was centrifuged, the semen plasma discarded, and the sperm pellet rediluted with a Tris-glucose-egg yolk extender containing 3% glycerol (Ext-1) at a sperm concentration of 200 x 10(6) spermatozoa (spz)/ml. The diluted semen was divided in three aliquots of equal volume and allowed to equilibrate for 1h at 4 degrees C. After equilibration, the same volume of three different second extenders was added, respectively, to each of the three aliquots: (A) Ext-2A (same composition as Ext-1 except that it contained 7% glycerol and 1% Equex STM Paste), (B) Ext-2B (same composition as that of Ext-1 except that it contained 7% glycerol and 1% Equex Pasta), and (C) Ext-2 (CONTROL: same composition as that of Ext-1 except that it contained 7% glycerol). Semen samples were packed in 0.5 ml straws and frozen on a rack 4 cm above liquid nitrogen (LN(2)) in a styrofoam box. Thawing was at 70 degrees C for 8s. Sperm motility was evaluated after thawing and at 1 h intervals for 5h at 38 degrees C by subjective examination and by using a CASA system. Plasma membrane integrity and acrosomal status were evaluated at 1, 4 and 7h post-thaw using a triple staining procedure and flow cytometry. Intracellular Ca(2+) concentration of live spermatozoa was evaluated by flow cytometry at 1, 4 and 7h post-thaw after co-loading the sperm cells with the Ca(2+) indicators Fluo 3 AM and Fura Red AM, and with PI. Post-thaw sperm survival and longevity, as well as the quality of the sperm movement, were significantly better (P<0.005) when Ext-2A (containing Equex STM Paste) was used. There was no difference between Ext-2B (containing Equex Pasta) and Ext-2 (CONTROL). The mean intracellular Ca(2+) concentration (arbitrary units) of cryopreserved spermatozoa (range: 0.23+/-0.12 to 1.26+/-0.46) was higher than that of fresh spermatozoa (0.13+/-0.06). When using Ext-2A, the live spermatozoa frequently (P=0.012) appeared divided in two subpopulations, with high (1.26+/-0.46) and low (0.27+/-0.09) intracellular Ca(2+) content, respectively. When using Ext-2B or Ext-2, the live spermatozoa were more frequently seen in a single population with low intracellular Ca(2+) concentration (0.30+/-0.35 and 0.23+/-0.12, for Ext-2B and Ext-2, respectively).

Effect of different glycerol treatments on frozen-thawed dog sperm longevity and acrosomal integrity

Four different concentrations of glycerol in a Tris-fructose-citric acid extender for frozen dog semen and the effects of adding glycerol at 37 degrees C or 4 degrees C to the extender were studied by monitoring the post-thaw sperm longevity and acrosomal integrity during incubation at 39 degrees C. In the first part of this study, ejaculates from 13 dogs were pooled and divided into 4 aliquots, which were centrifuged and the sperm pellets rediluted with a Tris-fructose-citric acid extender containing 2, 4, 6 and 8% (v/v) glycerol, respectively. Progressive motility by subjective estimation, live:dead spermatozoa ratio using eosin-nigrosin staining, and acrosomal integrity using phase contrast microscopy were evaluated before processing and at 0, 0.5, 1, 2 and 4 hours post-thawing incubating the semen samples in the dark at 39 degrees C. The experiment was performed using seven replicates and it was found that sperm motility and acrosomal integrity were superior following the use of 8% glycerol in the extender. In Experiment 2, 13 ejaculates from the same dogs used in the first experiment were pooled and divided into 3 aliquots, and an 8% glycerol diluent was added at 37 degrees C and 4 degrees C after 1 h of cooling or at 4 degrees C after 2 h of cooling, respectively. After freezing and thawing the same parameters as studied in the first experiment were assessed. The experiment was performed in 7 replicates, and no difference was found between treatments.

Viability assessment of dog spermatozoa using flow cytometry

The percentages of living and dead spermatozoa in fresh dog semen samples were assessed by means of a dual staining technique using carboxifluorescein diacetate (CFDA) and propidium iodide (PI). Two ejaculates were obtained from dogs, each ejaculate was divided into 4 aliquots, and different proportions of freeze-killed cells were added to each aliquot. Data obtained by flow cytometry analysis of each sample were compared with those obtained by the microscopic evaluation under epifluorescence illumination and by phase-contrast microscopy evaluation of the samples stained with eosin-nigrosin. Regression analysis was used to compare the 3 methods for membrane integrity assessment of canine spermatozoa, and high correlation coefficients were found between the flow cytometry procedure and the 2 microscopy techniques. The results from this study validate the use of flow cytometry as a precise method for assessing the viability of dog spermatozoa.

Effects of cryopreservation on the motile sperm subpopulations in semen from Asturiana de los Valles bulls

The aim of this study was to identify different motile sperm subpopulations in ejaculates from an autochthonous bull breed (Bos taurus) and to determine possible modifications in these subpopulations resulting from cryopreservation. Ejaculates were collected and cryopreserved following a conventional protocol. The overall sperm motility and the kinematic parameters of individual spermatozoa were evaluated in fresh ejaculates, after 4h at 5 degrees C, and at 0 and 2h postthaw. A multivariate clustering procedure separated 23,585 motile spermatozoa into four subpopulations: Subpopulation 1 showed medium velocity (VCL: 99.4+/-17.8 microm/sec) and high progressiveness (LIN: 65.1+/-14.0%); Subpopulation 2 included spermatozoa with high velocity (VCL: 148.7+/-25.6 microm/sec) but a nonprogressive trajectory (LIN: 33.1+/-10.5%); Subpopulation 3 represented slowly motile (VCL: 58.3+/-24.3 microm/sec) and nonprogressive sperm (LIN: 39.6+/-18.3%); and Subpopulation 4 included very rapid (VCL: 152.8+/-25.7 microm/sec) and highly progressive sperm (LIN: 70.9+/-13.7%). Subpopulation 4 was present in the greatest quantity in fresh ejaculates (36%), but after cooling, it significantly decreased (21%) concomitantly with an increase (P<0.001) in Subpopulation 2 (from 21% in fresh to 34% in postcooled semen). After freezing and thawing, the overall sperm motility was reduced, mainly due to Subpopulation 2 decreasing from 34% after cooling to 14% after thawing. Differences among bulls in the frequency distribution of spermatozoa within subpopulations were evidenced after thawing by different proportions of spermatozoa in Subpopulations 2 and 4. The current results indicate that a structure of four sperm subpopulations may be a common characteristic of bovine ejaculates and that the cooling phase of cryopreservation seems to be the determinant of postthaw semen quality.

Use of Ultrasound in the Reproductive Management of Dairy Cattle

The significant decrease in fertility observed in dairy cattle during the last few decades and increasing requirements by the farmers have made a regular control of reproduction indispensable to urgently identify and solve potential problems affecting reproductive efficiency. Traditionally, the main diagnostic methods used for reproductive control in cattle included rectal palpation, inspection of vaginal discharge and vaginoscopy. Since the 1990 s, the use of ultrasound (US) has become a common diagnostic method as a result of the new advances made in the development of US scans: smaller size, high level of autonomy, high image quality and accessible prices. Ultrasound improves accuracy in the diagnoses of stages of the oestrous cycle, ovarian and uterine pathologies, and pregnancy diagnosis. In addition, it facilitates the diagnosis of alterations during pregnancy (embryo mortality, foetal malformations, etc.) and helps determining foetal sex from day 55 of pregnancy.

Evaluation of sperm subpopulation structure in relation to in vitro sperm-oocyte interaction of frozen-thawed semen from Holstein bulls.

The present study examined the relationship between the relative amount of high motile sperm and sperm-oocyte interactions obtained from Holstein bull ejaculates. Post-thaw sperm motility was analyzed using a computer-assisted sperm analyzer system and evaluated to determine the sperm motility subpopulations. Adhesion and penetration of zona pellucida (ZP) and pronucleus formation using post-thawed samples (15 ejaculates form 5 different bulls) with different percentages of sperm in the subpopulation with the fastest and most progressive subpopulation (subpopulation 4 [SP4]) were analyzed. The correlation between the proportion of sperm in SP4 and the number of spermatozoa bound to the zona pellucida (ZBA), the penetration rate, and the rate of pronucleus formation were calculated. A significant (P < 0.05) and positive correlation was found between the number of spermatozoa bound to the zona pellucida, the penetration rate, and the rate of pronucleus formation with the proportion of sperm in SP4 (r = 0.79, r = 0.66, and r = 0.63, respectively). Our results suggest that this specific high motile and progressive subpopulation is positively and significantly correlated with the ability of a thawed bull semen sample to interact properly with the oocyte and its extracellular vestments. These findings emphasize the relevance of analyzing semen subpopulation composition to predict bull sperm fertilizing ability and to select Holstein bulls for breeding purposes.

Motile sperm subpopulations in frozen-thawed dog semen: changes after incubation in capacitating conditions and relationship with sperm survival after osmotic stress.

In this study we investigated the changes that in vitro incubation under capacitating conditions could induce on the motile sperm subpopulations present in frozen-thawed dog semen samples. In addition, cryopreserved dog spermatozoa were exposed to CCM (canine capacitating medium) solutions of 300, 150, 100 and 75 mOsm and the proportions of live spermatozoa with swollen tails were recorded (HOST+). Finally, frozen-thawed dog semen samples were submitted to a second cycle of freezing and thawing and the overall sperm motility, as well as the motile sperm subpopulations structure, was determined. Cryopreserved dog semen samples were structured in four sperm subpopulations with different motility characteristics: Subpopulation (Sp) 1 contained moderately rapid and progressive spermatozoa (25.2 ± 8.5%), Sp 2 included poorly motile and non progressive sperm (15.3 ± 8.1%), Sp 3 was represented by moderately slow non progressive sperm (14.9 ± 5.9%), and Sp 4 contained the most rapid and progressive sperm (20.8 ± 14.7%). After 3h of incubation under capacitating conditions, percentages of spermatozoa assigned to Sp 2 (6.1 ± 3.4%) and 3 (4.9 ± 2.8%) significantly decreased, whereas those assigned to Sp 1 (17.0 ± 11.2%) and 4 (16.2 ± 12.8%) did not significantly change. Significant correlations were found between percentages of HOST+, for the 3 osmolarities tested, and percentages of spermatozoa included in Sp 1 and 4 after 3 h of incubation in capacitating conditions or in Sp 4 after double freezing and thawing. These results indicated that subpopulations with the most rapid and progressive sperm seemed to be highly resistant to in vitro incubation in capacitating conditions and to osmotic stress, suggesting they are likely to be the source of the fertilizing population.

Reproductive Performance of Rabbit Does Artificially Inseminated via Intravaginal Administration of [des‐Gly 10, d‐Ala6]‐LHRH Ethylamide as Ovulation Inductor

This study was aimed to evaluate the reproductive performance of rabbit does artificially inseminated (AI) with a GnRH analogue [des-Gly10, D-Ala6]-LHRH. ethylamide to induce ovulation by intravaginal administration, delivered in the seminal dose. In a preliminary experiment, 39 does were divided into three groups (n = 13) that, at the time of AI, received the following ovulation induction treatments: (i) control group: 20 microg of gonadorelin administered intramuscularly; (ii) 25 microg of the GnRH analogue added to the seminal dose; (iii) 30 microg of the GnRH analogue added to the seminal dose. Fertility did not differ between the three groups (control: 80.6%, group 2: 82.8%, group 3: 73.3%). In a second experiment, a large-scale field trial was conducted to test the use of 25 microg of the GnRH analogue [des-Gly10, D-Ala6]-LHRH ethylamide delivered in the seminal dose (n = 270) against 20 microg of gonadorelin administered intramuscularly. Fertility was higher (p < 0.05) when ovulation was induced by intravaginal administration of the GnRH agonist (91.1% vs 85.6%). Prolificacy or mortality at birth was never affected by the ovulation induction treatments. In a third experiment, two groups of does [control group (n = 39): ovulation was induced using 20 microg of gonadorelin administered intramuscularly; treatment group (n = 40): ovulation was induced using 25 microg of [(des-Gly10, D-Ala6)-LHRH ethylamide added to the seminal dose] were inseminated at 42-day intervals for five successive AI cycles, to test the response to the GnRH agonist after repeated intravaginal administration to the same animals. Fertility and prolificacy were not influenced by the ovulation induction treatment neither there was an interaction between treatment and parity. The last experiment was aimed to determine whether it could be possible to add the GnRH agonist to the semen in the AI Center, just after semen collection and dilution, or it would have to be added in the farm, immediately before AI. Kindling rates did not significantly differ when ovulation was induced by intramuscular injection of gonadorelin (84.5%) or when the GnRH agonist was added to the seminal dose just at the moment (93.8 %) or 24 h before AI (90.4 %), but it was significantly lower when the hormone was added to the semen 32 h before AI (76.3 %). Prolificacy, however, was not influenced by the ovulation induction treatment.

Benchmarking welfare indicators in 73 free-stall dairy farms in north-western Spain

The aim of this study was to describe the status of body condition score (BCS), hock injuries prevalence, locomotion and body hygiene score as animal welfare measures in 73 free-stall dairy cattle farms in Lugo (Spain). A benchmarking process was established across farms: (1) the animal-based indicators were ordered from low to high values; (2) The farms were classified into three categories based on the number of indicators within less than the 25th percentile, 25th to 75th percentile and above the 75th percentile. The median prevalence of unsuitable BCS, hock injuries and clinical lameness was (median (range)) 51.7 per cent (13.3 to 89.5 per cent), 40.0 per cent (7.0per cent to 100 per cent) and 9.0 per cent (0per cent to 60.0 per cent) respectively. The dirtiness of the cow’s coat had a high prevalence (73.0 per cent (37.5per cent to 100 per cent)). Most farms did not display consistently good or poor animal-based indicators and each farm had its own set of strong and weak points. Moreover, facilities design and management practices were described to understand source of the observations made of the cows. The incidence of overstocking was 31.5 per cent for stalls and 26.0 per cent for headlocks. The front lunge space was reduced (<90 cm) on most dairies (90.4 per cent). Signs of poor natural ventilation (cobwebs or humidity on the roof) and ammonia odour were observed on 32.8 per cent and 85.0 per cent of the barns totally closed or with a side openingless than 50 per cent of the wall height. The milking parlour was designed with two or more turns more than 90° (9.3 per cent), and failed to allow cows to see the parlour before entering (45.2 per cent). On 52.0 per cent of dairies, more than 15 per cent of the cows had to be forcefully moved into the milking parlour. In conclusion, there was a big variation in the animal welfare levels within and across farms and they could benefit from others by changing management practices related to facilities and herds.