Microdroplet In Vitro Fertilization Can Reduce the Number of Spermatozoa Necessary for Fertilizing Oocytes

Successful in vitro fertilization (IVF) in mice has been achieved using spermatozoa at concentrations specifically optimized for the experimental conditions, such as species and source of spermatozoa. Although IVF in mice is mostly performed using about 80–500 µl drops, it is expected that the number of spermatozoa used for insemination can be reduced by decreasing the size of the IVF drops. The present study was undertaken to examine the extent to which the number of spermatozoa used for IVF could be reduced by using small droplets (1 µl). We devised the experimental parameters using frozen–thawed spermatozoa from C57BL/6 mice in anticipation of broader applications to other mouse facilities. We found that as few as 5 spermatozoa per droplet could fertilize oocytes (1 or 3 oocytes per droplet), although the fertilization rates were low (13–15%). Practical fertilization rates (> 40%) could be achieved with frozen-thawed C57BL/6J spermatozoa, which are sensitive to cryopreservation, when 20 sperm per droplet were used to inseminate 3 oocytes. Even with spermatozoa from a very poor quality suspension (10% motility), about 25% of oocytes were fertilized. Our calculations indicate that the number of inseminated spermatozoa per oocyte can be reduced to 1/96–1/240 by this method. In two separate embryo transfer experiments, 60% and 47%, respectively, of embryos developed to term. Our microdroplet IVF method may be particularly advantageous when only a limited number of motile spermatozoa are available because of inadequate freezing-thawing or genetic reasons.     

On the Species Specificity of Sperm Binding and Sperm Penetration of the Zona Pellucida

Sperm binding and sperm penetration of the zona pellucida (zp) are regarded as species‐specific. In this investigation, the interactions between bovine oocytes and porcine, respectively, equine spermatozoa have been studied under in vitro conditions and compared with the normal in vitro fertilization of bovine oocytes by bovine sperm. Surprisingly, many of the heterologous spermatozoa adhered firmly to the bovine oocytes and could not be removed by intense washing. On average, more than 100 boar or equine spermatozoa were bound to the zp of bovine oocytes. Electron microscopic studies clearly demonstrated that porcine sperm attached to the zona and underwent the acrosome reaction. Equine spermatozoa displayed a similar binding affinity, but unlike the porcine spermatozoa even penetrated the zp and were taken up into the oocyte after a longer period of co‐incubation. Considering these new results the dogma of a strict species specificity of sperm zona interactions under in vitro conditions has to be reconsidered.     

Effects of Melatonin Implants During Non‐Breeding Season on Sperm Motility and Reproductive Parameters in Rasa Aragonesa Rams

The effect of melatonin implants administered during non‐breeding season in Rasa Aragonesa rams on sperm motility parameters and other reproductive traits was assessed. In a first experiment, two Rasa Aragonesa rams were implanted (with melatonin group M), remaining other two males as control group (C). Semen of each group was collected from 1 May to 23 June, twice or three times a week, and motility parameters were assessed using a computer‐assisted sperm analysis system. Melatonin increased the percentage of progressive motile spermatozoa, particularly during 46–75 days after melatonin implantation (p < 0.01). In experiment 2, M and C in vitro fertilization ability had been determined by zona‐pellucida binding assays, using spermatozoa from experiment 1, obtained 60–70 days after melatonin was implanted. A significantly higher number of spermatozoa attached per oocyte was observed in frozen‐thawed immature ovine oocytes incubated with sperm from M animals than in those incubated with sperm from the C group (p < 0.01). Finally, a field assay (experiment 3) was performed. In this case, five Rasa Aragonesa rams were implanted with melatonin and three remained as control group. Sperm doses from those animals were used for artificial insemination of 2608 Rasa Aragonesa ewes from 39 different farms at non‐breeding season. Fertility, litter size and fecundity were studied. Semen from melatonin implanted rams seemed to increase both fertility and fecundity in ewes inseminated with spermatozoa obtained 46–60 days after implantation (p < 0.1). Thus, melatonin treatment in rams during non‐breeding season modifies sperm motility parameters and seems to improve the fertilization parameters obtained.     

Semen quality and fertility after heat stress in boars

Sperm morphology and the fertilizing capacity of ejaculated spermatozoa were examined in 6 Swedish Landrace boars before and after heat stress. The boars were exposed to 35° C during 100 h in a climatic room. Fertility was measured by insemination of gilts before and at various times after heat stress. Each gilt (n = 44) was inseminated with a total of 5×10⁹ spermatozoa diluted to 10O ml with EDTA-glucose diluent and fertilization was assessed by examining recovered ova 2 days after insemination.Changes in semen quality varied among the boars from a very weak response in 2 boars to pronounced semen alterations occurring 2–6 weeks after heat stress in the other boars. A close relationship was found between seminal changes and fertilization rates, all ejaculates which had high fertilization rates being of the same quality as the pre-exposure ejaculates. The ejaculates that had poor fertility were characterized by lowered sperm motility and increased numbers of spermatozoa with abnormal heads, proximal cytoplasmic droplets and nuclear pouch formations.     

Morphological features of spermatozoa of swamp buffalo AI bulls in Thailand

The appearance and incidence of sperm abnormalities was studied in 115 ejaculates, collected periodically over 1 year covering all seasons from five mature, healthy swamp buffalo (Bubalus bubalis) bulls reared under tropical conditions and serving as the current source of semen for artificial insemination (AI) in Thailand. Light microscopy of stained smears was used to investigate sperm head shape morphology, while unstained wet smears were used to examine other sperm abnormalities. The most commonly found morphological aberrations were pear-shaped spermatozoa, knobbed acrosomes, proximal cytoplasmic droplets, simple bent tails and coiled tails under the head, whose ultrastructure (scanning electron microscopy) corresponded to what has been found in other species of bovidae, including varieties of buffalo. The mean prevalence (as least squares mean +/- SEM) of sperm abnormalities was low (below 15%), corresponding to healthy spermiograms. The younger bulls (<10 years old, n = 3) had less abnormalities than the older ones (10.1 +/- 0.6% versus 14.1 +/- 0.8%, P < 0.001, n = 2), including abnormalities of sperm head shape (1.1 +/- 0.3% versus 3.6 +/- 0.3, P < 0.001), acrosome defects with knobbed acrosomes (1.1 +/- 0.2% versus 1.2 +/- 0.3%, P < 0.001), spermatozoa with proximal cytoplasmic droplets (2.7 +/- 0.1% versus 1.4 +/- 0.2%, P < 0.001), defective mid-pieces (0.2 +/- 0.1% versus 0.3 +/- 0.1%) and abnormal sperm tails (3.1 +/- 0.3% versus 5.7 +/- 0.4%, P < 0.001). The within-bull effect of the year solely affected the incidence of pear-shaped spermatozoa while the incidences of abnormal contour, variable size of sperm head shapes, abnormal mid-piece and simple bent tail among bulls were affected by ejaculate (week of collection). Interaction between age and ejaculate affected only the prevalence of spermatozoa with proximal cytoplasmic droplets. In conclusion, the types of defects encountered were similar to those found in other bovidae, with a very low prevalence over the year the AI sires were followed through.     

In vitro Capacitation and Acrosome Reaction of Dog Spermatozoa can be Feasibly Attained in a Defined Medium Without Glucose

Incubation of dog spermatozoa in a medium without glucose and in the presence of lactate and pyruvate (l-CCM) for 4 h at 38.5 degrees C in a 5% CO(2) atmosphere induced in vitro capacitation of these cells. This was verified after the combined specific capacitation-like changes in percentages of viability and altered acrosomes, motility characteristics, sperm location of reactivity against Pisum sativum, Arachis hypogaea and Helix pomatia lectins and the tyrosine phosphorylation pattern. Furthermore, a feasible acrosome reaction (AR) was induced when spermatozoa incubated in l-CCM for 4 h were further co-incubated for 1 h with canine oocytes. This was demonstrated by AR-like changes in percentages of viability, altered acrosomes, motility characteristics and sperm location of reactivity against P. sativum, A. hypogaea and H. pomatia lectins. All these results clearly indicate that in vitro capacitation, and subsequent AR, can be feasibly achieved without the presence of sugars. This ability can be related to the specific characteristics of energy-metabolism regulation reported in dog spermatozoa.     

Morphology and Chromatin Integrity of Stallion Spermatozoa Prepared by Density Gradient and Single Layer Centrifugation Through Silica Colloids

The objective was to investigate whether it is possible to improve the quality of stallion semen, with respect to sperm morphology and chromatin integrity, both of which have been linked to fertility, using either density gradient centrifugation (DGC) or a new method, hereby named single layer centrifugation (SLC). The two methods of colloidal centrifugation were evaluated using 38 ejaculates from 10 stallions. Sperm morphology, subjective motility and sperm chromatin integrity were compared in uncentrifuged samples and in centrifuged sperm preparations. The proportion of morphologically normal spermatozoa varied between stallions (p < 0.001) and was increased by both methods of colloidal centrifugation (median value before centrifugation 67.5%; after SLC 78%; after DGC 77%; p < 0.001). The incidence of certain abnormalities was reduced, e.g. proximal cytoplasmic droplets were reduced from 12.9% to 8.8% (p < 0.001), and mid-piece defects from 5.3% to 1.4% (p < 0.05). Similarly, sperm motility and chromatin integrity were significantly improved (p < 0.001), with no difference between the two centrifugation methods. Centrifugation through colloids can enrich the proportions of stallion spermatozoa with normal morphology and normal chromatin structure in sperm preparations. The new method, SLC, was as effective as DGC in selecting motile stallion spermatozoa with normal morphology and intact chromatin. SLC, being simpler to use than DGC, would be appropriate for routine use by stud personnel to improve stallion sperm quality in insemination doses.     

Morphological and acrosomal changes of canine spermatozoa during epididymal transit

Background

During epididymal transit, functional and structural modifications leading to full maturation enable male gametes to reach, recognize and fertilize the oocytes. In dogs, little is known on the modifications of spermatozoa during the passage in the epididymis. The aim of this study was to describe the motility, morphology and acrosomal patterns of canine spermatozoa retrieved from the epididymis caput, corpus and cauda.

Results

After the dilution required for the collection of epididymal content, sperm motility was significantly higher (P <0.0001) in the cauda compared to corpus and caput.Proportions of spermatozoa with normal morphology were significantly higher in corpus (P =0.02) and cauda (P <0.0001) compared to caput. Overall morphological abnormalities of the head and neck/midpiece were similar in the three different epididymal regions. A significantly increased prevalence of tail defects, mainly represented by single bent tails, was observed in the corpus compared to caput (P <0.0001) and cauda (P =0.006).Numbers of immature sperm with cytoplasmic droplets decreased from the proximal to the distal region of the epididymis. Particularly, proximal cytoplasmic droplets were more frequently found in spermatozoa collected from the caput epididymis than in the corpus (P <0.0001) and in the cauda (P <0.0001), whereas the occurrence of distal cytoplasmic droplets was higher in the corpus than in the caput (P =0.0003) and in the cauda (P <0.05).Significantly higher proportions of spermatozoa with intact acrosomes were retrieved from the cauda epididymis than from the caput (P =0.03) and the corpus (P =0.008). This difference was mainly due to a lower proportion of spermatozoa with abnormal acrosomes (mainly swollen acrosomes) rather than with absent acrosomes.

Conclusions

Canine spermatozoa undergo several modifications in the epididymis. The acquisition of progressive motility, migration of the cytoplasmic droplet and acrosomal reshaping lead to mature spermatozoa which are then stored in the cauda epididymis. From this site, spermatozoa can be retrieved and used in assisted reproductive techniques as a valuable tool for propagating genetic traits of high value individuals that dies accidentally or undergoes orchiectomy for medical purposes. Further investigations should be also focused on the potential use of spermatozoa recovered from other epididymal regions.     

The effect of Mycoplasma bovis on fertilization processes in vitro with bull spermatozoa and zona-free hamster oocytes

The effect of Mycoplasma bovis (Donetta strain) on the ability of bull spermatozoa to interact with zona pellucida-free hamster oocytes was studied in an in vitro assay. Ejaculates of semen from a fertile Holstein bull were used fresh on the day of collection (unextended semen) as well as diluted with egg yolk-citrate and used the following day (extended semen). The addition of M. bovis to both unextended and extended semen at a mycoplasma to sperm cell ratio of 10:1 significantly reduced sperm penetration rates and the mean number of sperm per penetrated egg. Similarly, the ability of spermatozoa to form pronuclei and the activation of penetrated oocytes were adversely affected by M. bovis. No apparent effect on sperm motility was detected. When M. bovis was added to the oocytes, there was a marked reduction in the sperm penetration rates and fertilization processes suggesting that the organism affects certain oocyte function(s). The results indicate that the presence of M. bovis in semen or in the female reproductive tract may affect fertilization. Moreover, the in vitro assay with hamster oocytes was found to be useful for demonstrating the effects of contaminating microbial agents on bovine fertilization processes.     

Sperm motility, plasma membrane integrity, and binding capacity to homologous zona pellucida of cryopreserved epididymal spermatozoa in the domestic cat

We examined motility, plasma membrane integrity, and binding capacity to homologous zona pellucidae (ZP) of frozen/thawed epididymal cat sperm as a model species for endangered felines. Epididymal spermatozoa from 20 domestic cats were frozen with freezing egg-yolk extender containing 3.0% glycerol in 0.25-ml straws. Post-thaw motility and plasma membrane integrity of the frozen/thawed spermatozoa were 31.8 +/- 2.4% and 32.2 +/- 4.2%, respectively. The frozen/thawed spermatozoa were co-cultured with frozen/thawed immature homologous oocytes with intact ZP for 3 h to examine their ability to bind to the ZP. Sixteen of the 20 frozen/thawed sperm samples demonstrated the ability to bind to ZP. These results indicated that the freezing system for epididymal sperm used in the present study gives appropriate information for banking the genetic resources of wild felid species.     

Quality and Fertilizing Ability In Vivo of Sex‐Sorted Stallion Spermatozoa

Little information is available on the quality of stallion spermatozoa after sex sorting. The objectives of the present study were to assess the quality of sex‐sorted stallion spermatozoa and determine its fertilizing ability after hysteroscopic low dose insemination. Ejaculates from four stallions were collected and sorted by a MoFlo SX^® flow cytometer/sperm sorter. Before and after sorting, spermatozoa were evaluated for motility by Computer Assisted Sperm Analysis, viability (SYBR 14‐propidium iodide), mitochondrial function (JC‐1) and acrosomal status (fluorescein isothiocyanate Pisum sativum agglutinin conjugated). A fertility trial was carried out on four mares (seven oestrous cycles) by hysteroscopic insemination, depositing 5 × 10⁶ X‐bearing spermatozoa. Sex sorting resulted in a significant decrease (p < 0.001) in all motility characteristics. Sperm viability and percentage of spermatozoa with functional mitochondria were not affected by the sorting process, while the percentage of reacted spermatozoa was higher (p < 0.01) for non‐sorted than sorted spermatozoa. Pregnancy rate was 28.6% (2/7) after low dose hysteroscopic insemination. Only one pregnancy was carried to term with the birth of a healthy filly. In conclusion, despite the reduction in sperm motility, sex sorting did not impair stallion sperm viability and mitochondrial activity immediately post‐thaw; moreover, the sexed spermatozoa retained the ability to fertilize in vivo.     

Birth of Kids After Artificial Insemination with Sex‐Sorted,Frozen‐Thawed Goat Spermatozoa

Successful sex‐sorting of goat spermatozoa and subsequent birth of pre‐sexed kids have yet to be reported. As such, a series of experiments were conducted to develop protocols for sperm‐sorting (using a modified flow cytometer, MoFlo SX^®) and cryopreservation of goat spermatozoa. Saanen goat spermatozoa (n = 2 males) were (i) collected into Salamon's or Tris catch media post‐sorting and (ii) frozen in Tris–citrate–glucose media supplemented with 5, 10 or 20% egg yolk in (iii) 0.25 ml pellets on dry ice or 0.25 ml straws in a controlled‐rate freezer. Post‐sort and post‐thaw sperm quality were assessed by motility (CASA), viability and acrosome integrity (PI/FITC‐PNA). Sex‐sorted goat spermatozoa frozen in pellets displayed significantly higher post‐thaw motility and viability than spermatozoa frozen in straws. Catch media and differing egg yolk concentration had no effect on the sperm parameters tested. The in vitro and in vivo fertility of sex‐sorted goat spermatozoa produced with this optimum protocol were then tested by means of a heterologous ova binding assay and intrauterine artificial insemination of Saanen goat does, respectively. Sex‐sorted goat spermatozoa bound to sheep ova zona pellucidae in similar numbers (p > 0.05) to non‐sorted goat spermatozoa, non‐sorted ram spermatozoa and sex‐sorted ram spermatozoa. Following intrauterine artificial insemination with sex‐sorted spermatozoa, 38% (5/13) of does kidded with 83% (3/5) of kids being of the expected sex. Does inseminated with non‐sorted spermatozoa achieved a 50% (3/6) kidding rate and a sex ratio of 3 : 1 (F : M). This study demonstrates for the first time that goat spermatozoa can be sex‐sorted by flow cytometry, successfully frozen and used to produce pre‐sexed kids.     

Consequences for the bovine embryo of being derived from a spermatozoon subjected to post-ejaculatory aging and heat shock: development to the blastocyst stage and sex ratio

The objective was to determine whether aging of sperm caused by incubation at normothermic (38.5 C) or heat shock (40 C) temperatures for 4 h prior to oocyte insemination affects sperm motility, fertilizing ability, competence of the resultant embryo to develop to the blastocyst stage and blastocyst sex ratio. In the first experiment, the percent of sperm that were motile was reduced by aging (P<0.001) and the reduction in motility was greater for sperm at 40 C compared to sperm at 38.5 C (P<0.01). In the second experiment, oocytes were inseminated with aged sperm. A smaller percent of oocytes fertilized with sperm aged at either temperature cleaved by Day 3 after insemination than oocytes fertilized with fresh sperm (P<0.05). There was no effect of sperm aging on the percent of oocytes or cleaved embryos that developed to the blastocyst stage. Aging of sperm before fertilization at 38.5 C reduced the percent of blastocysts that were male (P=0.08). In the third experiment, incubation of sperm at 38.5 C or 40 C for 4 h did not reduce fertilizing ability of sperm as determined by pronuclear formation at 18 h post insemination. In conclusion, aging of sperm reduced cleavage rate and the percent of blastocysts that were males but had no effect on the developmental capacity of the embryo. The effect of aging on cleavage rate may represent reduced motility and errors occurring after fertilization and pronuclear formation. Aging at a temperature characteristic of maternal hyperthermia had little additional effect except that polyspermy was reduced. Results indicate that embryo competence for development to the blastocyst stage is independent of sperm damage as a result of aging for 4 h at normothermic or hyperthermic temperatures.     

Consequences for the bovine embryo of being derived from a spermatozoon subjected to oxidative stress

Objective To determine whether oxidative damage of ejaculated frozen–thawed sperm prior to oocyte insemination in vitro affects the competence of the resultant embryo to develop to the blastocyst stage. Method Extended frozen semen from bulls was thawed, subjected to Percoll gradient purification to obtain motile spermatozoa and mixed with medium containing the pro-oxidants menadione or tert-butyl hydroperoxide. After 3 h at 38.5°C, the sperm were washed and used to inseminate oocytes in vitro. Embryo development proceeded until 8 days after insemination. Results Treatment of sperm with 15 or 30 µmol/L menadione reduced the proportions of oocytes that cleaved and those that developed to the blastocyst stage; 30 µmol/L menadione reduced the proportion of cleaved embryos that developed to the blastocyst stage at day 8 after insemination. Oocytes inseminated with sperm treated with 150 or 300 µmol/L tert-butyl hydroperoxide had lower proportions of cleavage and blastocyst development, and the proportion of cleaved embryos becoming blastocysts was also reduced. Conclusion Oxidative damage to ejaculated sperm can compromise the ability of the sperm to cause oocyte cleavage and leads to formation of embryos with reduced competence for development.     

A Scanning Electron Microscopy Study of Frozen / Thawed Dog Sperm During In Vitro Gamete Interaction

The aim of this work was to study the effects of cryopreservation on the binding and penetration of dog spermatozoa to the zona pellucida (ZP) by scanning electron microscopy (SEM). The sperm-rich fraction of six ejaculates from five dogs was divided into two aliquots and washed by centrifugation. One aliquot was processed as fresh control sample and the other aliquot frozen in Tris-fructose extender. Gamete interaction was assessed using in vitro matured bitch oocytes, which were co-incubated for up to 3 h. At hourly intervals after the start of co-incubation, in vitro fertilized (IVF) oocytes were processed by SEM. The results were analysed statistically using the anova test. Differences in binding and penetration of the spermatozoa to the ZP occurred; a lower proportion of oocytes with spermatozoa bound to ZP was observed using frozen sperm (p < 0.05) than with fresh sperm (61%, 57% and 53% vs 42%, 40% and 44% at 1, 2 and 3 h, respectively). The percentage of ZP penetration by fresh sperm was directly proportional to the time of co-incubation (9%, 25% and 34%; p < 0.05); in contrast, no differences were observed in the penetration rate with frozen-thawed sperm (21%, 17% and 21%). More acrosome reacted sperm were observed in frozen sperm than in fresh sperm on the surface of the ZP. The differences in the percentage of binding and penetration between fresh and frozen sperm during the co-culture could indicate that the time course of penetration is faster in frozen-thawed dog spermatozoa than in fresh sperm, but that fresh spermatozoa can penetrate more oocytes over a given period of time, which may be related to their reacted or non-reacted initial status.     

Function of the Cumulus Oophorus Before and During Mammalian Fertilization

Fertilization encompasses a series of different steps which have to be performed in a well‐orchestrated way to create a new individual. They include sperm capacitation, sperm binding and penetration of the zona pellucida, traversing the perivitelline space, binding and fusion with the oolemma, activation of the oocyte and decondensation of the sperm head to form the male pronucleus. In most mammalian species, cumulus cells surround the oocyte at the time of fertilization. Removal of the cumulus oophorus at this point of time often leads to a drop in fertilization rates. It is not yet known how cumulus cells interact with the oocyte or with spermatozoa to promote fertilization. There are different possibilities:

• 1 cumulus cells cause mechanical entrapment of spermatozoa and guide hyperactivated spermatozoa towards the oocyte, while preventing abnormal spermatozoa to enter the cumulus matrix;
• 2 cumulus cells create a micro‐environment for the spermatozoa which favours their capacitation and penetration into the oocyte;
• 3 cumulus cells prevent changes in the oocyte which are unfavourable for normal fertilization; these changes can be located in the zona pellucida or in the cytoplasm.

In this review, studies in several species are listed to prove the importance of these three cumulus cell functions and the current lines of research are highlighted. Moreover, different ways to improve in vitro fertilization of bovine cumulus‐denuded oocytes are discussed.     

A spermatozoal midpiece abnormality associated with infertility in a Llasa Apso dog

The semen of a 9-year-old Llasa Apso dog with a recent history of infertility was examined by light and electron microscopy. The presenting sperm abnormality was a high percentage (87 per cent) of cells with proximal cytoplasmic droplets. With the higher resolution of differential interference contrast microscopy and with transmission electron microscopy (TEM) it was apparent that not all of the midpiece abnormalities had the appearance of typical cytoplasmic droplets. Furthermore, by TEM both axonemal and mitochondrial defects were readily demonstrated, often with evidence of degenerative mitochondria in cytoplasmic swellings at other levels of the midpiece. These severe abnormalities were apparent only by TEM of thin sections, although scanning EM was useful in identifying abnormal midpiece outlines. It is suggested that the failure of droplet migration and the midpiece ultrastructural defects were linked to a common fault in spermateliosis.     

In Vitro Sperm Penetration Speed and its Relationship with In Vivo Bull Fertility

The in vitro tests utilized to evaluate sperm quality represent an interesting and important approach to evaluate ejaculated fecundant capacity. In the present work, the oocyte-penetrating ability of sperm from two bulls with low and two with high in vivo fertility rate was investigated. Sperm-quality parameters, such as sperm concentration, total and progressive motility, acrosome and total sperm anomalies, proximal cytoplasmic drops and live : dead sperm ratio were assessed and batches of sperm homologues in these parameters were selected. In expt 1, a sperm : oocyte ratio of 3000 was used and the oocytes were fixed 5, 10, 15 and 18 h post-insemination (hpi). In expt 2, the sperm : oocyte ratio was reduced to 1000 and the eggs were fixed at 8 and 10 hpi. The results, analysed by chi-square test, showed significant differences (p <0.001) among bulls at 15 hpi in expt 1 and 8 hpi in expt 2; nevertheless, no accordance was found between sperm penetration rate and in vivo fertility. At 18 hpi, the low-fertility bulls exceeded the two high-fertility bulls, supporting previous reports that suggest an opposite correlation between in vivo and in vitro fertility rate at low doses of heparin. Furthermore, a more efficient zona binding ability of one high-fertility bull was pointed out in expt 2 after the reduction of sperm : oocyte ratio, as it reached the highest percentage of penetration when compared with all the others.     

Studies on the Intracellular Ca2+ Concentration of Frozen–Thawed Dog Spermatozoa: Influence of Equex from Different Sources, Two Thawing Diluents and Post-thaw Incubation in Capacitating Conditions

The addition of 0.5% (v/v) of Equex STM Paste (Nova Chemical Sales, Scituate Inc., MA, USA), whose active ingredient is sodium dodecyl sulphate (SDS), to a Tris–egg yolk extender was demonstrated to improve the longevity of frozen–thawed dog spermatozoa during in vitro incubation at 38°C. The aim of the first experiment was to compare the effects of two SDS‐containing compounds, Equex STM Paste and Equex Pasta (Minitüb, Tiefenbach, Germany), when added to a Tris–egg yolk based extender, on the post‐thaw longevity of dog spermatozoa, as well as on the intracellular Ca²⁺ concentration of spermatozoa, during post‐thaw incubation at 38°C. The post‐thaw sperm survival and longevity, as well as the quality of the sperm movement, were significantly better when using Equex STM Paste. Such prolonged sperm longevity, however, was associated to a higher intracellular Ca²⁺ concentration in a large subpopulation of the live spermatozoa. A second experiment was aimed to evaluate the effects of sperm dilution immediately post‐thaw with a Tris buffer containing glucose or fructose. The two Tris buffers were no different for any of the sperm parameters studied. The aim of a third experiment was to evaluate the sperm longevity, motility patterns and intracellular Ca²⁺ concentration of cryopreserved dog spermatozoa during post‐thaw incubation in capacitating conditions [canine capacitating medium (CCM) with or without 5 μg/ml of heparin]. Heparin had no significant effects on any of the sperm parameters evaluated. During the first 8 h of incubation, the majority of the live spermatozoa had a high intracellular Ca²⁺ content. However, after 8–10 h of incubation, it had significantly declined. The highest proportion of fast motile sperm, and the highest curvilinear velocity, average path velocity and amplitude of lateral head displacement for the total motile sperm were observed during the 2–4‐h incubation period. It was concluded that: (a) the addition of 0.5% (v/v) of Equex STM Paste to a Tris–egg yolk based extender significantly improved the post‐thaw longevity of dog spermatozoa, but the same concentration of Equex Pasta had no significant beneficial effects; (b) sperm dilution after thawing with a Tris buffer containing glucose or fructose made no difference in post‐thaw sperm longevity; (c) the addition of 5 μg/ml of heparin to CCM had no significant capacitating effects on frozen–thawed dog spermatozoa.     

Heterologous In Vitro Fertility Evaluation of Cryopreserved Iberian Red Deer Epididymal Spermatozoa with Zona-intact Sheep Oocytes and its Relationship with the Characteristics of Thawed Spermatozoa

A heterologous in vitro system, using zona‐intact sheep oocytes, was used to evaluate the relationship between sperm factors of Iberian red deer thawed epididymal sperm and the percentage of cleaved oocytes. Epididymal spermatozoa were recovered from six males, diluted with freezing extender and cryopreserved. After thawing sperm motility (SM) and acrosome and membrane integrities were evaluated. Again, these parameters were assessed after incubation in freezing extender at 37°C for 2 h. After cryopreservation the values for SM and acrosome and membrane integrities were high (～80, 80 and 70% respectively). However, these values significantly decreased after incubation (～59, 62 and 47% respectively). Red deer thawed epididymal sperm fertilized zona‐intact sheep oocytes, although the percentage of cleaved oocytes was low (～22%). No relationship was found between sperm parameters assessed after thawing and the percentage of cleaved oocytes. Likewise, any sperm parameter evaluated after incubation was assessed in relation to the percentage of cleaved oocytes. However, acrosome and membrane integrities were near to significance (p = 0.06 and p = 0.09 respectively). Then, we conducted a reduced model with these two variables and both were related to the percentage of cleaved oocytes (p = 0.02 and p = 0.04 respectively). Thus, acrosome and membrane integrities were related to the percentage of cleaved oocytes negatively and positively respectively. It was concluded that the classical parameters assessed in deer thawed sperm samples can be good predictors of the ability to fertilize zona‐intact sheep oocytes.