Postpartum deaths: piglet, placental, and umbilical characteristics.

The fetal growth of the piglet is highly dependent on its placenta, and the newborn piglet birth weight is highly associated with postpartum death. However, there is little information available in the literature on the assessment of the placenta in relation to postpartum death in piglets. The aim of this study was to evaluate the impact of the placental area and placental weight, status of the umbilical cord, and piglet birth characteristics, such as blood parameters, vitality score, and birth weight on postpartum death. All live born piglets in litters from 26 Landrace-Yorkshire sows were monitored during farrowing and the status of each was recorded, including placental area and placental weight and blood variables obtained from the piglets and umbilical veins. Out of the 386 live-born piglets, 16.8% died before weaning at 5 wk. Among these, 78.5% died within the first 3 d of life. Mean blood concentration of lactate was increased in piglets that did not survive to weaning (P = 0.003). Concentrations of hemoglobin and hematocrit were decreased (P < 0.001) compared with survivors. Piglets born with a broken umbilical cord had a reduced vitality score vs. piglets born with an intact umbilical cord (P = 0.021), and they had an increased probability of dying before weaning (P = 0.050). Mean birth weight, body mass index, placental area (P < 0.001), and placental weight (P = 0.020) were reduced in piglets that died before weaning vs. those that survived. Birth weight and placental area were furthermore negatively associated with live litter size. Blood concentrations of IgG and albumin recorded at d 1 were decreased in piglets that died before weaning (P < 0.01), and blood concentration of albumin was positively associated with placental area (P < 0.001). We conclude that placental area and placental weight, status of the umbilical cord, birth weight, body mass index, blood concentrations of lactate, hemoglobin, and hematocrit recorded at birth, and blood concentrations of IgG and albumin recorded at d 1 were associated with postpartum death in this study. These results may indicate that there is an upper uterine limitation of litter size and that placental area and placental weight influence postpartum survival.

The relationship between post-thaw sperm DNA integrity and non-return rate among Norwegian cross-bred rams.

With the aim of investigating the relationship between sperm DNA integrity and non-return rate (NRR) among Norwegian cross-bred rams, semen from 15 individuals was examined by flow cytometry. Sperm Chromatin Structure Assay (SCSA) quantifies the proportion of spermatozoa with denatured DNA after in situ acid treatment, and the four parameters % DFI, % HDS, MEAN DFI and SD DFI are all different measures of DNA denaturation and maturation. Field fertility, reported as NRR 25 days after insemination was based on all inseminations from a large-scale breeding programme and supplied by the Norwegian Association of Sheep and Goat Farmers. From each ram, four straws from four different weeks of the breeding season were analysed, and the associations between 25-day NRR and the mean of the four SCSA parameters were tested using a logistic regression model. The results revealed no association between fertility and % DFI or % HDS, while SD DFI and MEAN DFI showed a significant negative association with NRR. Further, the SCSA values varied significantly between ejaculates within ram among some of the rams in the study. However, no significant association was seen between these intra-individual differences in sperm DNA integrity and NRR. In conclusion, this study suggests an association between sperm DNA integrity and NRR for rams. However, further research must be conducted to confirm these findings and determine whether sperm DNA assessments can be applied to predict ram fertility.

Associations between intrapartum death and piglet, placental, and umbilical characteristics.

Intrapartum death in multiparous gestations in sows (Sus scrofa) is often caused by hypoxia. There is little information in the literature on the assessment of the placenta in relation to intrapartum death in piglets. The aim of this study was to evaluate the impact of the placental area and weight upon piglet birth characteristics and intrapartum death. Litters from 26 Landrace-Yorkshire sows were monitored during farrowing and the status of each piglet was recorded, including blood parameters of piglets and their umbilical veins. Of 413 piglets born, 6.5% were stillborn. Blood concentrations of glucose, lactate, and CO(2) partial pressure were increased in the stillborn piglets (P < 0.05) and corresponding umbilical veins (P < 0.01) vs. live-born piglets, whereas pH and base excess were decreased (P < 0.001). Time from onset of parturition until birth was increased for piglets born dead vs. live (P < 0.001). Mean birth weight for piglets born dead was not different from live-born piglets (P = 0.631), whereas mean body mass index was reduced (P < 0.001). Mean placental area and placental weight belonging to stillborn piglets were not different from live-born piglets (P = 0.662 and P = 0.253, respectively). Blood concentrations of lactate, hemoglobin, and hematocrit recorded in all piglets pooled were associated with placental area (P < 0.05), but not with placental weight (P > 0.2). Piglet BW was positively correlated with placental area and placental weight (P < 0.001). The risk of being born dead increased with increasing birth order group, and broken umbilical cords explained 71% of the stillbirths (P = 0.001). We conclude that placental area and placental weight are both positively associated with piglet birth weight, but not with the probability of being born dead. Placental area was a better predictor of piglet vitality than placental weight. Because umbilical cord rupture and prolonged birth time were associated with being born dead, umbilical cord rupture and placental detachment seem to be probable causes of intrapartum death.

Blood variables and body weight gain on the first day of life in crossbred pigs and importance for survival.

Improving survival is a continuous objective in swine breeding. The aim of this study was to record 22 blood variables and BW gain on the first day of life in Landrace-Yorkshire-Duroc crossbred piglets and to find associations between these variables and survival at weaning. All live piglets from 18 litters were weighed and blood sampled at birth and on d 1 and were monitored to weaning at the age of 5 wk. A total of 261 piglets were born, of which 8.8% were stillborn. Additionally, 15.1% died before weaning. The blood variables glucose, immunoglobulins, and white blood cells increased from birth to d 1 (P < 0.001), whereas α(1)- and ß(1)-globulin, red blood cells, hemoglobin, and hematocrit decreased (P < 0.001). At birth, concentrations of lactate (P = 0.004), pH (P = 0.007), red blood cells (P = 0.017), hemoglobin (P = 0.018), and hematocrit (P = 0.052) were associated with survival to weaning. Also, concentrations of lactate increased (P = 0.030) and pH decreased (P < 0.001) when piglets were born in the last third of a litter. On d 1, concentrations of glucose (P = 0.015), hemoglobin (P = 0.025), and BW gain (P = 0.001) were all decreased in piglets that did not survive to weaning. Body weight gain also decreased (P = 0.005) when piglets were born in the last third of a litter. Concentrations of IgG on d 1 was not associated with survival at weaning (P = 0.230) but decreased (P < 0.001) when piglets were born in the last third of a litter. We conclude that several blood variables recorded at birth and on d 1 and BW gain on d 1 were highly associated with survival at weaning and that piglets born in the last third of the litter had less favorable vitality.

Customizing semen preservation protocols for individual dogs and individual species: sperm preservation beyond the state of the art.

Sperm quality can be variable in morphometric and physiological attributes between males of different species, between males within species subtypes reared under different environmental conditions, between ejaculates of the same male or even between sperm populations within an ejaculate. Clinical semen evaluation is based on evaluation of whole ejaculates, which is not a chemically or physiologically well-defined entity, rather a collection of heterogeneous subpopulations giving different measurements and possessing different fertilizing potential. Identification of subpopulations with different motility patterns is important as well as characterizing the subtle structural changes underlying the motility differences observed. The ability to identify populations of sperm responding rapidly or failing to progress through the capacitation process may have clinical applications. Studies of lipid-phase fluidity of sperm membranes, mathematical modelling of membrane ion transport, role of modifying components and detergent-resistant microdomains are of particular interest. When customizing extenders to ejaculates from cryosensitive males or species, a thorough knowledge of species sperm membrane physiology and an assessment of the individual ejaculate's sperm populations are necessary. Structural differences have been found in sperm membranes between fox species with different cryosurvival potential of their spermatozoa. Supplementation of lipids and detergents in cryoextenders may influence membrane fluidity of the surviving spermatozoa in a species-dependent manner and influence capacitation. Immobilization of sperm prior to cryopreservation with subsequent slow release of sperm in the female genital tract may be a way to prolong the fertile life of sperm. In canids with a long oocyte maturation time, delayed capacitation may be beneficial.

Vaginal deposition of frozen-thawed semen in Norwegian Dairy goats: comparison of single and double insemination with equal total number of spermatozoa.

In a field trial, a total of 472 Norwegian Dairy goats showing natural estrus were artificially inseminated with frozen-thawed semen. The farmers themselves performed vaginal deposition of 400 x 10(6) spermatozoa; one half of the does received two straws (200 x 10(6) spermatozoa/straw) at the same time (single AI), while the other half received two straws (200 x 10(6) spermatozoa/straw) 12 h apart (double AI). The commercially available extender Andromed was used for dilution. The does were housed at 15 different farms, and on average 31 does were inseminated per farm. Non return rates (NRR) and kidding rates after single insemination were 64.3% and 58.3%, respectively. Double inseminations resulted in a NRR of 62% and a kidding rate of 57%. No significant difference between single and double AI was seen in the study. This study indicates that single or double vaginal insemination with an equal total number of frozen-thawed spermatozoa (400 x 10(6)) can give acceptable fertility results in Norwegian Dairy goats. However, studies on reducing sperm numbers are called for to allow AI donor bucks to be used to their fullest potential.

Alterations of sperm DNA integrity during cryopreservation procedure and in vitro incubation of bull semen.

An association between sperm DNA integrity and fertility was recently shown for frozen-thawed Norwegian Red (NRF) bull semen diluted in skimmed milk egg yolk (SMEY). In general the fertility of NRF cattle is high, however, in comparison with NRF semen in SMEY, NRF semen diluted in Tris EY based extenders has shown reduced fertility. The aim of the present study was to do a split-sample comparison of sperm DNA integrity of NRF bull semen (n=20) in SMEY and Triladyl (Tris EY based) during routine cryopreservation procedure and during in vitro incubation of frozen-thawed semen in modified synthetic oviduct fluid (mSOF). In contrast to the high fertility of NRF cattle, Holstein cattle are experiencing a marked decline in fertility. Therefore, the present study also aimed to compare sperm DNA integrity of NRF (n=20) and Holstein (n=20) semen diluted in Triladyl during in vitro incubation. The sperm DNA integrity was measured by susceptibility to in situ acid induced denaturation by the Sperm chromatin structure assay (SCSA). Compared to initial values of frozen neat semen, an increase in DNA damage was observed after dilution and cooling (5 degrees C) and after freezing-thawing of NRF semen in SMEY, but only after freezing-thawing for NRF semen diluted in Triladyl. Sperm DNA damage of NRF semen increased during in vitro incubation in mSOF; the increase in percentage of spermatozoa with DNA damage was more prominent in SMEY than in Triladyl, while the degree of damage was higher in Triladyl, throughout the incubation period. However, while the correlation between DNA damage and sperm survival was negative in SMEY throughout the incubation period, a positive correlation was observed in Triladyl after 9h of incubation, indicating a higher presence of DNA damage in the live sperm population. In comparison with Holstein spermatozoa, the sperm DNA integrity of NRF semen reflected a better ability to withstand alterations induced during in vitro incubation in mSOF. In conclusion, sperm DNA integrity of NRF bull semen was altered during the cryopreservation procedure and in vitro incubation in mSOF. Dilution in Triladyl maintained bull sperm DNA integrity better than dilution in SMEY. Furthermore, alterations in Holstein sperm DNA integrity was more pronounced during in vitro incubation in mSOF compared to NRF bull spermatozoa.

Cryopreservation of canine semen - new challenges.

Egg yolk (EY) protects cell membranes against cold shock, and it prevents or restores the loss of phospholipids from the membrane. EY has been widely used in semen extenders. It has been added to Tris-Glucose buffer and has been widely used for cooling and cryopreservation of canine semen. EY is not a defined entity, but a complex biological compound containing proteins, vitamins, phospholipids, glucose and antioxidants which are all potentially useful for cell membrane integrity. Unfortunately, it also is a biologically hazardous compound. Hence, whole EY needs to be replaced by other chemically defined components for semen processing in dogs. Freezing poor semen does not improve its quality, so attention must be focused on how to cope with dogs whose semen does not freeze well, and on designing individual freezing extenders for semen from such males. Furthermore, differences have been found among canid species in the ability of their spermatozoa to withstand freezing. There are differences in sperm membrane fatty acid composition among species, which may explain part of these differences. If the presence of long-chained polyunsaturated fatty acids contributes to increased membrane fluidity, this relationship may be biphasic, i.e. either too much membrane fluidity, or too little, could compromise successful sperm cryopreservation. An increase in fluidity of the outer leaflet of the plasma membrane has been shown in frozen thawed dog spermatozoa. The protective effect of exogenous lipids may lie in close association with the membrane rather than in modification or rearrangement of the membrane. This also points at lipids as an important, if not entirely new group of substances, which may substitute standard EY-based diluents in preserving sperm survival during freezing. EY-derived phospholipids or lecithin could be used to replace whole EY. Vegetable lecithin is currently investigated to avoid using substances of animal origin. EY also contains antioxidants which prevent cells from oxidative damage due to the generation of reactive oxygen species. An increasing number of publications now recognize the significance of protecting sperm from this damage during processing by using dietary or diluent supplemented antioxidants. This paper aims at looking at some of the new challenges in freezing of dog semen.

Artificial insemination in canids: a useful tool in breeding and conservation.

Artificial insemination (AI) and semen freezing have become services available to dog owners worldwide, and the demand for services to freeze semen is increasing. In other canids such as the fox, the fur industry utilizes fresh or frozen semen to artificially inseminate vixens to produce pelts. Clearly, AI facilitates the use of a male to sire several females by diluting the ejaculate, increases breeding hygiene, and allows crossing between species with slightly different breeding seasons. The African wild dog (Lycaon pictus) is currently considered by the World Conservation Union (IUCN) as one of most endangered canids. In captive populations of African wild dogs, semen has been frozen with encouraging results, using a standard cryopreservation protocol for domestic dogs, but successful AI has not been reported. In wolves, there is one report regarding the live birth of an offspring after intravaginal AI of a deslorelin-induced estrous female. In 2005, three Mexican gray wolf females were artificially bred by intrauterine insemination with freshly collected semen from unrelated males, and all females whelped. Artificial insemination may be vaginal, intrauterine or intratubal, and the semen may be fresh, fresh and chilled (diluted), or frozen-thawed, and the source of semen may be epididymal or ejaculated. In the domestic dog, the results are good to excellent for AI with all three types of processed semen when the source is ejaculated semen, whereas epididymal sperm still yields poorer results. Species differences in female physiology, as well as differences in the cryotolerance of the sperm from various canid species, warrant further research and development.

Sperm DNA damage is related to field fertility of semen from young Norwegian Red bulls.

Flow cytometry was utilised for the first time to independently measure five sperm parameters of individual spermatozoa of bull ejaculates to differentiate between outcome successes after artificial insemination (AI). These parameters included plasma membrane and acrosome integrity, mitochondrial functionality and DNA damage measured by sperm chromatin structure assay (SCSA) and terminal deoxynucleotide transferase-mediated dUTP nick end labelling (TUNEL) assays. For each parameter, results of 142 ejaculates (30 bulls) were ranked into three groups according to their flow cytometric measures: (1) ejaculates with the 25% lowest measures; (2) the 50% middle measures; and (3) the 25% highest measures. In total, 20 272 first-service inseminations (18 ;10(6) spermatozoa per AI dose) were performed, where fertility was defined as non-return within 60 days after first insemination. While plasma membrane and acrosome integrity, and mitochondrial functionality were not significantly related to fertility, data from SCSA and TUNEL assays were significantly associated with fertility. Ejaculates in SCSA group 1 had higher odds of AI success (1.07, 95% CI = 1.02-1.12), whereas those in group 3 had lower odds of AI success (0.94, 95% CI = 0.89-0.99), compared with the average odds of all three groups. Ejaculates in group 2 did not have significantly higher odds of AI success compared with the average odds. For TUNEL-positive spermatozoa, the odds of AI success was higher in group 1 compared with the average odds (1.10, 95% CI = 1.02-1.13), whereas odds of AI success in groups 2 and 3 were not significant compared with the average odds. In conclusion, despite the high number of spermatozoa per AI dose from high-quality bulls, both SCSA and TUNEL assays were valuable measures in this study for evaluating sperm quality in relation to fertility after AI.

Artificial insemination with frozen semen in dogs: a retrospective study of 10 years using a non-surgical approach.

From 1994 to 2003, a total of 526 bitches of 99 different breeds were artificially inseminated in 685 estrus cycles with domestic (n = 353) or imported (n = 332) frozen-thawed semen from 368 males. The overall whelping rate was 73.1% and mean (+/- S.E.M.) litter size 5.7 +/- 0.1 pups. The whelping rate was higher after intrauterine insemination (75.0%; n = 665) than after intravaginal insemination (10.0%, n = 20; P < 0.05). Insemination at the optimal time resulted in a higher whelping rate (78%, n = 559; P < 0.01) and larger litter size (5.8 +/- 0.2; P < 0.05) than inseminations performed late or too late (55.7% and 4.5 +/- 0.5, n = 61). Two inseminations (n = 384) yielded a higher whelping rate (P < 0.05) and mean litter size (P < 0.01) than one insemination (n = 241), 78.1% and 6.0 +/- 0.2 and 70.5% and 5.1 +/- 0.2, respectively. For inseminations performed at the optimal time, however, the whelping rate was not significantly different for bitches inseminated twice (79.3%, n = 358) versus once (76.8%, n = 168), but the litter size was larger (6.0 +/- 0.2 and 5.3 +/- 0.3). Semen classified as of poor quality (progressive motility < 50% or percentage abnormal sperm > 20%) resulted in a lower whelping rate (P < 0.01) than semen classified as of good quality (progressive motility > or = 50% and percentage abnormal sperm < or = 20%), 61 and 77%, respectively. Small breeds (n = 50) had a smaller litter size (3.9 +/- 0.3; P < 0.01) than larger breeds (medium [5.7 +/- 0.3, n = 94], large [5.9 +/- 0.2, n = 295] or giant breeds [6.1 +/- 0.5, n = 62] [P < 0.01]). Bitches older than 6 years had a lower whelping rate (68.2%) than younger ones (77.0%; P < 0.05). The duration of pregnancy was longer (P < 0.01) for bitches with a litter size of < 3 pups (61.7 +/- 0. 4 days, n = 30) than for bitches with larger litters (60.5 +/- 0.1 days, n = 177). These results show the potential of transcervical intrauterine insemination for routine artificial insemination in dogs. The results with frozen semen inseminations were optimised by inseminating bitches < or = 6 years old 2 and 3 days after ovulation with semen of good quality from males < or = 8 years old.

Comparative aspects of sperm membrane fatty acid composition in silver (Vulpes vulpes) and blue (Alopex lagopus) foxes, and their relationship to cell cryopreservation.

Cryogenic protocols have been developed for the storage of farmed silver fox (Vulpes vulpes) spermatozoa. However, these same protocols and modifications of these protocols have failed to satisfactorily preserve spermatozoa collected from farmed blue foxes (Alopex lagopus). Because cryogenic success has been linked to membrane composition, the plasma membrane lipid composition of farmed blue fox and silver fox spermatozoa was studied. Silver fox spermatozoal membranes have significantly higher levels of docosapentaenoic acid (DPA; 22:5, n-6) compared to blue fox spermatozoa, and blue fox spermatozoal membranes have significantly higher levels of stearic acid (18:0). Silver fox spermatozoal membranes not only have a higher ratio of unsaturated/saturated membrane fatty acids, but also higher levels of membrane desmosterol and cholesterol.

Mapping of the bovine genes of the de novo AMP synthesis pathway.

Summary The purine nucleotides adenosine monophosphate (AMP) and guanosine monophosphate (GMP) are critical for energy metabolism, cell signalling and cell reproduction. Despite their essential function, little is known about the regulation and in vivo expression pattern of the genes involved in the de novo purine synthesis pathway. The complete coding region of the bovine phosphoribosylaminoimidazole carboxylase gene (PAICS), which catalyses steps 6 and 7 of the de novo purine biosynthesis pathway, as well as bovine genomic sequences of the six other genes in the pathway producing inosine monophosphate (IMP) and AMP [phosphoribosyl pyrophosphate amidotransferase (PPAT), phosphoribosylglycinamide formyltransferase (GART), phosphoribosylformylglycinamidine synthase (PFAS), adenylosuccinate lyase (ADSL), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) and adenylosuccinate synthase (ADSS)], were identified. The genes were mapped to segments of six different bovine chromosomes using a radiation hybrid (RH) cell panel. The gene PPAT, coding for the presumed rate-limiting enzyme of the purine de novo pathway was closely linked to PAICS on BTA6. These, and the other bovine locations i.e. GART at BTA1, PFAS at BTA19, ADSL at BTA5, ATIC at BTA2 and ADSS at BTA16, are in agreement with published comparative maps of cattle and man. PAICS and PPAT genes are known to be closely linked in human, rat and chicken. Previously, an expressed sequence fragment of PAICS (Bos taurus corpus luteum, BTCL9) was mapped to BTA13. By isolation and characterization of a BAC clone, we have now identified a PAICS processed pseudogene sequence (psiPAICS) on BTA13. Processed pseudogene sequences of PAICS and other genes of the purine biosynthesis pathway were identified in several mammalian species, indicating that the genes of this pathway have been susceptible to retrotransposition. The seven bovine genes are expressed at a higher level in testicular and ovary tissues compared with skeletal muscle.

Effects of in vitro storage time and semen-extender on membrane quality of boar sperm assessed by flow cytometry.

The Norwegian AI company Norsvin has used the short-term semen-extender BTS to extend and store boar semen since the late 1980s. Fertility results have been consistent when extended semen has been used for AI within 3 days after collection, however, from a production and economic point of view it is preferable that semen stored for up to 5 days can be used. The aim of this study was to compare membrane quality of sperm stored in BTS for 3 days with sperm stored in the long-term semen-extenders Androstar, Mulberry III and X-cell for 5 days. Using a split-sample design, plasma membrane- and acrosome-integrity were assessed flow cytometrically by use of Yo-Pro-1 and PNA-FITC, and fluidity and phospholipid asymmetry of the membrane were assessed by use of MC540 and Annexin V-FITC. Due to observed sperm fragmentation in Androstar after Day 1, the data for Androstar were excluded from the analyses. After 5 days of storage, the membrane quality of X-cell-stored sperm was not statistically different from that of sperm stored in BTS for 3 days, while membrane quality of sperm stored in Mulberry III was statistically better on Day 5 compared to BTS on Day 3. In conclusion, Mulberry III and X-cell preserve sperm quality, as well as that of BTS on Day 3, for up to 5 days after collection.

Components of litter size in mice after 110 generations of selection.

The aim of the present study was to evaluate how ovulation rate and survival rate through pregnancy had been affected by more than 110 generations of upwards selection on litter size in mice. The mean number of pups born alive was 22 in the high line (selected line) and 11 in the control line (an increase in 2.6 standard deviations). Selection on litter size increased ovulation rate by 4.6 standard deviations, and it is suggested that selection also increased embryonic mortality in late pregnancy. Embryo survival from ovulation until birth was 66% in the selected line and 69% in the control line, and the observed loss in litter size from day 16 of pregnancy until birth was possibly higher in the high line compared with the control line. Selection for higher litter size has significantly increased body weight in both males and females, as the mean weight at mating for the females was 46 g in the high line and 33 g in the control line respectively.

Seasonal changes in spermatogenic activity and in plasma levels of FSH, LH and testosterone, and the effect of immunization against inhibin in the male silver fox (Vulpes vulpes).

The cellular composition of the silver fox testis assessed by DNA flow cytometry and histological analysis exhibited marked circannual alterations. The proportion of haploid cells increased from late October to the breeding season in February, while that of diploid cells decreased and that of tetraploid cells fluctuated during the same period. Towards the end of March these changes were reversed. The seasonal variations in testicular histology paralleled the changes in distribution of cells from the different DNA populations. In August, 69% of the tubules contained spermatogonia as the only type of germ cell, while the remaining 31% also contained a few primary spermatocytes. In late October more than 50% of the tubules contained spermatocytes, and during the period of further activation from early December-February the seminiferous epithelium included round and/or elongated spermatids as well. In February, all tubules contained complete associations of germ cells, whereas in late March tubules with spermatogonia only and spermatogonia together with a few spermatocytes reappeared. In May, only such tubules could be found indicating total regression. Plasma concentrations of FSH and LH increased from early November, both gonadotrophins reaching maximum levels in December or early January, and then both declined during the second part of January, immediately prior to the actual breeding season. LH values showed a few smaller peaks in the beginning of June, whereas FSH levels were generally low until the next period of testicular reactivation. Testosterone concentrations were also low during most of the year but rose in November and December to reach a peak in January and a second peak in June. In animals immunized against inhibin the distribution of haploid, diploid and tetraploid cells did not deviate to any great extent from that in the controls, except in March when the immunized males had a markedly lower proportion of tetraploid cells, and in May, when they had a distinctly higher proportion of haploid cells. These findings were partly reflected by the histology. In the immunized animals, plasma FSH levels started to increase at approximately the same time but peaked higher and remained elevated almost 1 month longer than in the controls, whereas both the rise and decline in LH levels generally coincided with the variations in these animals, but the values were mostly higher. The testosterone profiles were similar to those in the controls except that the maximum values were also usually higher.

Maturation and fertilization of blue fox (Alopex lagopus) oocytes in vitro.

The aim of the present study was to assess the progression of meiotic maturation and fertilization of in vitro matured blue fox (Alopex lagopus) oocytes at different time intervals after in vitro maturation and insemination by the use of confocal laser scanning microscopy. A total of 242 immature oocytes from ovarian follicles 1-2 mm in diameter from seven ovulating blue fox vixens at oestrus were cultured for 48 h in TCM-199 before in vitro insemination with 5.0 x 10(5) frozen-thawed fox spermatozoa. Oocytes were transferred to 50 microliters microdroplets of modified Tyrode's medium without glucose at pH 7.7 under mineral oil, inseminated and cultured at 38 degrees C in a humidified atmosphere with 5% CO2 for 2 (n = 10), 4 (n = 22), 8 (n = 41), 20 (n = 52), 24 (n = 10), 30 (n = 48) and 48 h (n = 59). The oocytes and zygotes were stained with propidium iodide and were analysed by confocal laser scanning microscopy or epifluorescence microscopy. Only 125 of 242 oocytes (52%) could be evaluated: of these germinal vesicle breakdown (GVBD) was observed in five of nine oocytes (56%) at 2 h, eight of 18 oocytes (44%) at 4 h, eight of 18 oocytes (44%) at 8 h, 27 of 42 oocytes (64%) at 20-24 h, 14 of 19 oocytes (74%) at 30 h, and 13 of 19 oocytes (68%) at 48 h after insemination. In total, 75 of 125 oocytes (60%) underwent GVBD. All stages from the germinal vesicle to the four-cell stage embryos were observed, but the rate of cleavage was low (9%). Immature oocytes collected from small subordinate ovarian follicles of oestrous vixens after ovulation of the dominant follicles were able to mature and be fertilized, as well as undergo the first two cleavage divisions in vitro.

Artificial insemination with frozen semen in dogs: a retrospective study.

In a retrospective study, from 1994 to 1998, of inseminations with frozen semen in dogs, a total of 312 bitches of 70 different breeds were inseminated with imported (n = 183) or domestic (n = 129) semen. The overall whelping rate was 70% and mean (+/- SEM) litter size was 5.3 +/- 0.2 pups. The whelping rate was higher after intrauterine insemination (71%; n = 305) than after intravaginal insemination (29%; n = 7; P < 0.05). Timing of insemination was crucial; timing classified as optimal resulted in a higher whelping rate and larger litter size (P < 0.05) than did timing classified as early, late or too late. In the too late category, none of the bitches (n = 5) whelped. For optimal timing, whelping rate and mean (+/- SEM) litter size were 76% (n = 252) and 5.6 +/- 0.2, for early 33% (n = 6) and 1.5 +/- 0.5, and for late 47% (n = 19) and 2.8 +/- 0.7. Two inseminations yielded a higher whelping rate (P < 0.05) and greater mean litter size (P < 0.05) than that of one insemination, 77% and 5.6 +/- 0.3, and 60% and 4.6 +/- 0.3, respectively. However, the results obtained after one insemination were poorer partly because of an over-representation of late insemination in this group. Semen classified as of poor quality (progressive motility < 50% or percentage of abnormal spermatozoa > 20%) gave a lower whelping rate (53%) than did semen of medium (progressive motility = 50%) or good quality (progressive motility > 50% and percentage of abnormal spermatozoa < 20%), which gave whelping rates of 76 and 74%, respectively (P < 0.05). The mean litter sizes were not significantly different. Eighty-two per cent of bitches (120 of 147) inseminated twice into the uterus at a time classified as optimal with frozen semen of good or medium quality whelped. The mean (+/- SEM) litter size was 5.6 +/- 0.3 pups in this group. These results show the potential of transcervical intrauterine insemination for routine artificial insemination in dogs.

Current state in biotechnology in canine and feline reproduction.

Biotechnology has proceeded much further in cats than in canines, although the pregnancy rate after in vitro maturation (IVM), IVC and embryo transfer (ET) is still relatively low. The use of AI with frozen-thawed semen as a breeding tool to overcome breeding incompatibility or to preserve male genetic material has been limited in felines in contrast to the situation in domestic dogs and foxes. In many research scenarios and endangered felid species programs, the in vitro production of feline embryos with subsequent transfer has complemented the use of AI. Improvement of IVM, in vitro fertilization (IVF) and embryo culture coupled with ovarian tissue grafting, cryobanking of follicles, oocytes, semen, or embryos, with subsequent ET into surrogate females, may render this technology feasible for use in endangered wild felids. In canines, reliable systems for in vitro production of embryos, embryo cryopreservation and transfer are yet to be developed. The refinement of invasive fertilization techniques, such as intracytoplasmic sperm injection (ICSI), may eventually provide a tool for removal of recipient oocyte nuclei and transfer of selected embryonic or somatic cell donor nuclei into domestic cat ooplasm, thereby providing a tool for genetic modification, or for preservation of valuable genetic material.

Assisted reproductive technology in canid species.

Assisted reproductive technologies in dogs began as early as the 18th century. The first scientifically recorded artificial insemination (AI) was performed in Italy by Spallanzani and lead to the birth of three pups. Progress in the area was slow, and subsequent development included AI equipment and methods for short-term preservation of fresh, and later, for frozen semen which led to the world's first litter produced from frozen semen in 1969. Improvement of freezing methods and AI equipment from 1970 onwards has rendered AI useful as a breeding technique for dogs. In parallel, AI in foxes was developed in Scandinavia in the early 1980's; this resulted in the economically valuable crossbreeding of silver and blue foxes for the production of bluefrost pelts. Unfortunately, due to the particular physiology of the canine female, progress in other artificial breeding techniques has lagged behind. Only in the last few years have these techniques been successfully applied in basic research to study oocyte maturation, in vitro fertilization, embryo cryopreservation and embryo transfer in canids.