Hysteroscopic insemination of mares with low numbers of nonsorted or flow sorted spermatozoa

The objectives of this study were 1) to compare pregnancy rates resulting from 2 methods of insemination using low sperm numbers and 2) to compare pregnancy rates resulting from hysteroscopic insemination of 5 x 106 nonsorted and 5 x 106 spermatozoa sorted for X- and Y-chromosome-bearing populations (flow sorted). Semen was collected with an artificial vagina from 2 stallions of known acceptable fertility. Oestrus was synchronised (June to July) in 40 mares, age 3-10 years, by administering 10 ml altrenogest orally for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. All mares were given 3000 iu hCG i.v. at the time of insemination to induce ovulation. Mares were assigned randomly to 1 of 3 treatment groups: mares in Treatment 1 (n = 10) were inseminated with 5 x 10(6) spermatozoa deposited deep into the uterine horn with the aid of ultrasonography. Mares in Treatment 2 (n = 10) were inseminated with 5 x 10(6) spermatozoa deposited onto the uterotubal junction papilla via hysteroscopic insemination. Mares in Treatment 3 (n = 20) were inseminated using the hysteroscopic technique with 5 x 10(6) flow sorted spermatozoa. Spermatozoa were stained with Hoechst 33342 and sorted into X- and Y-chromosome-bearing populations based on DNA content using an SX MoFlo sperm sorter. Pregnancy was determined ultrasonographically at 16 days postovulation. Hysteroscopic insemination resulted in more pregnancies (5/10 = 50%) than did the ultrasound-guided technique (0/10 = 0%; P<0.05) when nonsorted sperm were inseminated. Pregnancy rates were not significantly lower (P>0.05) when hysteroscopic insemination was used for sorted (5/20 = 25%) and nonsorted spermatozoa (5/10 = 50%). Therefore, hysteroscopic insemination of low numbers of flow sorted stallion spermatozoa resulted in reasonable pregnancy rates.     

Quantitation of the fertilising ability of fresh compared with frozen and thawed fowl spermatozoa

The fertilising abilities of freshly-diluted and frozen and thawed fowl semen were compared by inseminating a wide range (0.2 to 1600 X 10(6] of doses of spermatozoa. These data demonstrated that there was a non-linear relationship between numbers of spermatozoa and the probability of fertilisation. It was concluded that a typical fertility trial, which assesses the percentage of fertile eggs laid by groups of hens inseminated with a fixed dose of semen, was inadequate for comparing the quality of semen samples. An alternative fertility trial is described, which involves assessing the numbers of spermatozoa required to fertilize a particular percentage of eggs laid. This modified method showed that the quality of frozen and thawed semen, in terms of its fertilising ability, was reduced to 1.6% of that of fresh semen.     

Morphological observations on frozen and thawed Muscovy spermatozoa

1. Fresh Muscovy drake spermatozoa were examined using a scanning electron microscope (SEM). The average lengths of the segments were: acrosome 1·8 μm, nucleus 10‐9 μm, midpiece 3·6 μm and flagellum (exclusive of midpiece) 71 μm.

2. Under the light microscope, the incidence of abnormal spermatozoa in Muscovy semen subjected to freezing and thawing (almost all with crooked necks) was about 5% higher than that in diluted unfrozen semen.

3. In thawed semen, various abnormalities of the acrosome were observed under the SEM. It seemed that the most radical change was the complete separation of the acrosome from the apical part of the nucleus.

4. The incidence of abnormal acrosomes was increased more than 20% by freezing and thawing.

5. These results suggest that low fertility in thawed semen may be related to increases in the proportion of spermatozoa with crooked necks and acrosomal damage.

     

Improved assessment of frozen/thawed mouse spermatozoa using fluorescence microscopy

Genetically modified (GM) animals are unique mutants with an enormous scientific potential. Cryopreservation of pre-implantation embryos or spermatozoa is a common approach for protecting these lines from being lost or to store them in a repository. A mutant line can be taken out of a breeding nucleus only if sufficient numbers of samples with an appropriate level of quality are cryopreserved. The quality of different donors within the same mouse line might be heterogeneous and the cryopreservation procedure might also be error-prone. However, only limited amounts of material are available for analysis. To improve the monitoring of frozen/thawed spermatozoa, commonly used in vitro fertilization (IVF) followed by embryo transfer were replaced with animal-free techniques. Major factors for assessing spermatozoa quality (i.e., density, viability, motility, and morphology) were evaluated by fluorescence microscopy. For this, a live/dead cell staining protocol requiring only small amounts of material was created. Membrane integrity was then examined as major parameter closely correlated with successful IVF. These complex analyses allow us to monitor frozen/thawed spermatozoa from GM mice using a relatively simple staining procedure. This approach leads to a reduction of animal experiments and contributes to the 3R principles (replacement, reduction and refinement of animal experiments).     

Birth of puppies of predetermined sex after artificial insemination with a low number of sex‐sorted,frozen–thawed spermatozoa in field conditions

The aim of this study was to evaluate fertility and sex ratios after artificial insemination in dogs under field conditions. Semen was cryopreserved as unsorted (control) or was separated into X‐ and Y‐chromosome‐bearing sperm using a cell sorter. Sixty female dogs were inseminated with frozen–thawed spermatozoa of 100 × 10⁶ unsorted (a dose in practice) and 4 × 10⁶ sorted (X and Y group, respectively). A total of 20 dogs became pregnant and 126 puppies were born from the three groups. The percentage of parturition was similar for the X (5/20; 25.0%) and Y (4/20; 20.0%) group (P > 0.05), but lower than controls (11/20; 55.0%) (P < 0.05). Ultimately 28 out of the 32 puppies produced from X group were female (87.5%) and 19/22 (86.4%) puppies of Y group were male. In contrast, sex ratio (51.4% to 48.6%) in the control was significantly different from the X, Y group (P < 0.05). However, male and female puppies in the control had similar birth weights and weaning weights to those from the X and Y groups. This preliminary information indicated that normal puppies of predicted sex can be produced with low numbers of sorted cryopreserved dog spermatozoa at a farm level, making sperm‐sexing technology potentially applicable for elite breeding units.     

Evaluation of membrane integrity of frozen/thawed deer spermatozoa: short communication

A simultaneous live/dead and acrosome staining, originally described for domestic mammals, was successfully applied on red deer (Cervus elaphus) and fallow deer (Dama dama) spermatozoa collected from the cauda epididymidis and vas deferens of shot stags. The staining is simple enough for routine application. Seven classes of spermatozoa were distinguished in the smears of frozen/thawed semen samples. Morphology, including cytoplasmic droplets, was evaluated as well. Percentage of live cells with intact acrosomes and with no other morphological aberrations might be a practical index of semen quality.     

Melatonin can improve viability and functional integrity of cooled and frozen/thawed rabbit spermatozoa

Melatonin is known to protect sperm against freezing-inflicted damage in different domestic species. The aim of the study was to evaluate the effect of supplementation of semen extender with melatonin on the quality and DNA integrity of cooled and frozen/thawed rabbit spermatozoa. We also investigated whether the addition of melatonin to the semen extender could improve the fertility of rabbit does artificially inseminated with frozen/thawed semen. Semen samples collected from eight rabbit bucks were pooled and then diluted in INRA-82 supplemented either with (0.5, 1.0 or 1.5 mM) or without (0.0 mM) melatonin. Diluted semen was cooled at 5°C for 24 hr. For cryopreservation and based on the first experiment's best result, semen samples were diluted in INRA-82 in the presence or absence of 1.0 mM melatonin and then frozen in 0.25 ml straws. Following cooling or thawing, sperm quality and DNA integrity were evaluated. Furthermore, the fertility of frozen/thawed semen was investigated after artificial insemination. Supplementation of semen extender with 1.0 mM melatonin improved (p < .05) motility, viability, membrane and acrosome integrities in cooled semen compared with other groups. Sperm quality and DNA integrity were higher (p < .05) in frozen/thawed semen diluted in 1.0 mM melatonin-supplemented extender than in the control group. Conception and birth rates were higher in does inseminated with 1.0 mM melatonin treated semen compared with the controls. In conclusion, supplementation of semen extender with 1.0 mM melatonin improved the quality of cooled and frozen/thawed rabbit spermatozoa. Melatonin can preserve DNA integrity and enhance the fertility of frozen/thawed rabbit spermatozoa.     

Artificial insemination with canine spermatozoa frozen in a skim milk/glucose-based extender

Due to the recent outbreak of avian influenza, transportation of frozen canine semen with egg yolk has been sharply restricted. Thus, there is urgent need to develop a novel egg yolk-free extender for freezing canine spermatozoa. In the present study, the effect of using skim milk/glucose (SG)-based extender without egg yolk on the motility and fertilizing capacity of canine spermatozoa frozen-thawed in the presence of glycerol was examined. There was a tendency for the proportion of motile spermatozoa exposed to SG-based extender for 3 h to be higher than that exposed for 1 h, but the difference was not significant. The motility and other viability parameters of canine spermatozoa after thawing were similar to those obtained with an egg yolk-based extender. When spermatozoa frozen with SG-based extender containing glycerol after 3 h exposure were transcervically inseminated into 2 recipient bitches, a total of 6 pups were obtained. These results suggest that a simple extender composed of skim milk, glucose and glycerol is useful for cryopreservation of canine spermatozoa, which may contribute to improved exchange of genetic material and efficient production of companion and working dogs, such as guide dogs for the blind.     

On the fertility and survival of deep frozen boar spermatozoa thawed in skim milk

Satisfactory conception rates of deep frozen boar spermatozoa were obtained, with insemination by way of the cervix, after thawing the deep frozen spermatozoa in boar seminal plasma, both in preliminary trials (Crabo & Einarsson 1971, Crabo et al. 1972 b) and in a large field trial (Einarsson et al. 1972). Fertility with pellet frozen boar spermatozoa, thawed without dilution, was reported by Graham et al. (1971 a, b) and Pursel & Johnson (1971).     

The effect of liposomes composed of phosphatidylserine and cholesterol on fertility rates using frozen thawed equine spermatozoa

The objective of this study was to determine if the addition of liposomes composed of phosphatidylserine (PS) and cholesterol (CH) to equine sperm would improve pregnancy rates after sperm were cryopreserved. Ejaculates from four stallions, collected every other day during May and June were split, treated with PSCH liposomes or HBS (Hepes Buffered Saline) and cryopreserved. Fifty-two mares were bred over eighty estrous cycles with the frozen-thawed semen. The one cycle pregnancy rates of the mares inseminated with semen treated with liposomes (45%) were similar to mares inseminated with control semen (48%; p>0.05). Thus, at least at the levels used, PSCH liposomes added to equine sperm did not improve pregnancy rates of mares inseminated with cryopreserved semen.     

Bitch fertility after natural mating and after artificial insemination with fresh or frozen semen

The birth rate and fecundity of 92 bitches of 32 different breeds mated naturally or following artificial insemination of fresh or frozen semen were compared. The birth rate after natural service was 92 per cent compared with 84 per cent when fresh semen was deposited in the uterine body. When frozen semen was inseminated intra-uterine a birth rate of 67 per cent was obtained whilst a figure of 25 per cent was obtained following intra-vaginal insemination of fresh semen. There were no differences in litter size.     

Ultrastructure of frozen and thawed ram sperm

With the aid of a transmission electron microscope we studied the ultrastructure of frozen and thawed ram sperm. The cryoprotective agents do not completely prevent the occurrence of structural changes in spermatozoa. The sperm membrane system is affected. The greatest and most frequent damage is to the acrosome, the cell membrane and mitochondria are less injured. We observed regional differences in the damage to the cell membrane. It is most damaged above the acrosome, least in the postacrosomal area and in the principal piece of the tail. We found considerable variability in the changes among individual spermatozoa from the same ejaculate. The changes are not in the nature of mechanical damage as a result of the occurrence of ice microcrystals. We therefore think that the occurrence of structural changes is dependent on damage at molecular level and consists of a change in the physical-chemical properties of membranes.     

Pregnancy rates in mares after a single fixed time hysteroscopic insemination of low numbers of frozen-thawed spermatozoa onto the uterotubal junction

REASONS FOR PERFORMING STUDY: To compensate for the wide variation in the freezability of stallion spermatozoa, it has become common veterinary practice to carry out repeated ultrasonography of the ovaries of oestrous mares in order to be able to inseminate them within 6-12 h of ovulation with a minimum of 300-500 x 10(6) frozen-thawed spermatozoa. Furthermore, in order to achieve satisfactory fertility, this requirement for relatively high numbers of spermatozoa currently limits our ability to exploit recently available artificial breeding technologies, such as sex-sorted semen, for which only 5-20 x 10(6) spermatozoa are available for insemination. OBJECTIVES: This study was designed to evaluate and compare the efficacy of hysteroscopic vs. conventional insemination when low numbers of spermatozoa are used at a single fixed time after administration of an ovulation-inducing agent. METHODS: In the present study, pregnancy rates were compared in 86 mares inseminated once only with low numbers of frozen-thawed spermatozoa (3-14 x 10(6)) at 32 h after treatment with human chorionic gonadotrophin (hCG), either conventionally into the body of the uterus or hysteroscopically by depositing a small volume of the inseminate directly onto the uterotubal papilla ipsilateral to the ovary containing the pre-ovulatory follicle. RESULTS: Pregnancy rates were similarly high in mares inseminated conventionally or hysteroscopically with 14 x 10(6) motile frozen-thawed spermatozoa (67% vs. 64%). However, when the insemination dose was reduced to 3 x 10(6) spermatozoa, the pregnancy rate was significantly higher in the mares inseminated hysteroscopically onto the uterotubal junction compared to those inseminated into the uterine body (47 vs. 15%, P < 0.05). CONCLUSIONS: When inseminating mares with <10 x 10(6) frozen-thawed stallion spermatozoa, hysteroscopic uterotubal junction deposition of the inseminate is the preferred method. POTENTIAL CLINICAL RELEVANCE: Satisfactory pregnancy rates are achievable after insemination of mares with frozen-thawed semen from fertile stallions 32 h after administration of human chorionic gonadotrophin (Chorulon). Furthermore, these results were obtained when mares were inseminated with 14 x 10(6) progressively motile frozen-thawed spermatozoa from 2 stallions of proven fertility.     

Comparative study of the effects of various cryoprotectants in preserving the morphology of frozen and thawed fowl spermatozoa

1. The effects of various cryoprotectants (glycerol, dimethyl‐sulphoxide (DMSO), methylformamide and ethylene glycol) of the same molarity on preserving the morphology of frozen and thawed fowl spermatozoa (especially midpiece and acrosome) were examined under the light (LM) and scanning electron microscope (SEM) to determine the most suitable.

2. Under LM, the mean respective increases in sperm deformity in semen diluted with ethylene glycol, methylformamide, glycerol and DMSO were 4·5, 4·9, 5·0 and 6·5 percentage points.

3. Under SEM, the mean respective increases in acrosomal deterioration in semen diluted with glycerol, ethylene glycol, DMSO and methylformamide were 6·6, 8·8, 13·1 and 31·2 percentage points.

4. From these results, it appears that glycerol is superior to ethylene glycol DMSO and methylformamide as a cryoprotectant.