Incidence and avoidance of zona fracture in cryopreserved bovine ova and embryos

Ova (n=62), which were collected from slaughterhouse bovine ovaries, and embryos (n=26), which were non-surgically recovered from 11 superovulated crossbred donor cows, were frozen. The frozen ova and embryos were then thawed using two conventional thawing protocols, i.e. at 37 °C for 30 seconds in a water bath and at 25 °C for 2 minutes in air. Some 64.5% of the ova and 53.8% of the embryos thawed in the water bath and 16.1% of the ova and 7.7% of the embryos thawed in ambient air exhibited fractured z:onae pellucidae. The slow thawing protocol had a lower incidence of zona damage in cryopreserved oval and embryos than the fast thawing protocol. A low pregnancy rate (12.5%) was recorded for embryos transferred with zona fracture while embryos transferred with intact zonae had a rate of 35.3%) indicating that embryos with zona damage are less viable.     

Interactions between straw size and thawing rates on the cryopreservation of agouti (Dasyprocta aguti) epididymal sperm

This study verifies the interactions between straw size and thawing rates and their impact on the epididymal sperm from this species. Caudae epididymidum from 10 agoutis were subjected to retrograde washing using a coconut water extender (ACP‐109c^®). Epididymal sperm were evaluated and extended in ACP‐109c^® plus egg yolk (20%) and glycerol (6%). The samples were packaged in 0.25‐ or 0.50‐ml straws, frozen in liquid nitrogen and thawed at 37°C/1 min or 70°C/8 s, followed by a re‐evaluation. The use of 0.25‐ml straws thawed at 37°C/1 min provided a value of 26.6% for sperm motility. No interactions between straw size and thawing rates were verified on agouti sperm (p > 0.05), but when 0.5‐ml straws were thawed at 70°C/8 s, sperm vigour decreased significantly (p < 0.05). It is recommended that the agouti epididymal sperm cryopreserved in ACP‐109c^® extender should be packaged in 0.25‐ or 0.50‐ml straws and thawed at 37°C/60 s.     

Migration and differentiation of gonadal germ cells under cross-sex germline chimeras condition in domestic chickens

A series of experiments was conducted to investigate migration, proliferation and differentiation of gonadal germ cells (GGCs) collected from the gonads of 7-day-old chick embryos under cross-sex germline chimera conditions. The migratory and proliferative abilities of exogenous GGCs were examined by transferring 50 fluorescently labeled GGCs collected from White Leghorn (WL) embryos into the blood of 2-day-old Rhode Island Red (RIR) embryos. No significant difference was observed in the number of fluorescently labeled GGCs in the gonads of recipient embryos among any of the four possible donor and recipient sex combinations. Cross-sex germline chimeras were produced to examine the differentiation of GGCs by transferring 100 GGCs from WL embryos into 2-day-old RIR embryos. Exogenous-GGC-derived progeny were obtained from both male and female recipients, except when female GGCs were transferred into male recipients. The migratory ability of GGCs recovered from the 7-day-old embryonic gonad was not influenced by cross-sex germ cell transfer conditions, whereas the differentiation of the GGCs was affected by the sex combinations of GGCs donors and recipients.     

Expression of Genes in the Canine Pre‐implantation Uterus and Embryo: Implications for an Active Role of the Embryo Before and During Invasion

The aim of the present study was to assess genes expressed in maternal uterine tissue and pre‐implantation embryos which are presumably involved in maternal recognition and establishment of canine pregnancy. For this purpose, 10 pregnant bitches were ovariohysterectomized between days 10 and 12 after mating. Four non‐pregnant bitches served as controls. Early pregnancy was verified by flushing the uterine horns with PBS solution. The collected embryos (n = 60) were stored deep‐frozen (?80°C). Uterine tissue was excised, snaps frozen in liquid nitrogen and homogenized using TRI Reagent. All embryos from one litter were thawed together and also homogenized in TRI Reagent. RT‐PCR was performed to prove mRNA expression of progesterone receptor, key enzymes of the prostaglandin synthesis pathway, selected growth factors, cytokines, immune cell receptors, major histocompatibility complex (MHC) and matrix‐metalloproteinases (MMP). Only pregnant uteri revealed the presence of mRNA for interferon (IFN)‐γ, IL‐4 and CD‐8, which resembles the milieu in humans and other mammalians. Similarly, in day 10 embryos, mRNA for transforming growth factor‐β, insulin‐like growth factor‐1,‐2, hepatocyte growth factor, leukaemia inhibitor factor, tumour necrosis factor‐α, interleukin‐1β,‐6,‐8, cyclooxygenase‐2, CD4⁺ cells, and MMP‐2 and ‐9 were detected, but not MHC‐I or ‐II. We therefore suppose that the canine embryo, like its human counterpart, actively initiates measures to prevent attacks from the maternal immune system to prepare its own adhesion, nidation, growth and further development.     

Effect of Cryopreservation on the Sperm DNA Fragmentation Dynamics of the Bottlenose Dolphin (Tursiops truncatus)

Sperm DNA fragmentation is one of the major causes of infertility; the sperm chromatin dispersion test (SCDt) evaluates this parameter and offers the advantage of species‐specific validated protocol and ease of use under field conditions. The main purpose of this study was to evaluate sperm DNA fragmentation dynamics in both fresh and post‐thaw bottlenose dolphin sperm using the SCDt following different cryopreservation protocols to gain new information about the post‐thaw differential sperm DNA longevity in this species. Fresh and cryopreserved semen samples from five bottlenose dolphins were examined for sperm DNA fragmentation dynamics using the SCDt (Halomax^®). Sperm DNA fragmentation was assessed immediately at collection and following cryopreservation (T0) and then after 0.5, 1, 4, 8, 24, 48 and 72 h incubation at 37°C. Serially collected ejaculates from four dolphins were frozen using different cryopreservation protocols in a TES‐TRIS‐fructose buffer (TTF), an egg‐yolk‐free vegetable lipid LP1 buffer (LP1) and human sperm preservation medium (HSPM). Fresh ejaculated spermatozoa initially showed low levels of DNA fragmentation for up to 48 h. Lower Sperm DNA fragmentation (SDF) was found in the second fresh ejaculate compared to the first when more than one sample was collected on the same day (p < 0.05); this difference was not apparent in any other seminal characteristic. While there was no difference observed in SDF between fresh and frozen–thawed sperm using the different cryopreservation protocols immediately after thawing (T0), frozen–thawed spermatozoa incubated at 37°C showed an increase in the rate of SDF after 24 h. Sperm frozen in the LP1^? buffer had higher levels (p < 0.05) of DNA fragmentation after 24‐ and 48‐h incubation than those frozen in TTF or HSPM. No correlation was found between any seminal characteristic and DNA fragmentation in either fresh and/or frozen–thawed samples.     

Cryopreservation and In Vitro culture of Preimplantation Embryos in Djungarian Hamster (Phodopus sungorus)

Although embryo cryobanking was applied to Syrian golden and to Campbell's hamsters, no attempt has been made at freezing embryos in Djungarian hamsters. Four‐cell stage embryos were flushed from the reproductive ducts of pregnant females before noon of the third‐day post coitum and frozen in 0.25‐ml straws according to standard procedures of slow cooling. A mixture of permeating (ethylene glycol) and non‐permeating (sucrose) cryoprotectants was used. The thawing was performed by incubating at RT for 40 s followed by 40 s in a water bath at 30.0°C. Most (66.7%) of the non‐frozen four‐cell embryos developed up to the morula stage in rat one‐cell embryo culture medium (R1ECM). The use of hamster embryo culture medium (HECM) yielded fewer morulas (18.2%) during the same 24‐h period of culture. The rate of embryo's surviving the freezing–thawing procedures, as estimated by light microscopy, was 60.7–68.8%. After 24‐h culturing in R1ECM, 64.7% of frozen–thawed four‐cell embryos developed and all of them reached the morula stage. Supplementation of R1ECM with GM‐CSF (2 ng/ml) improved the rate of Djungarian hamster frozen–thawed embryo development: 100% of the four‐cell stage embryos developed, 50% of them achieved the morula stage, and 50% developed even further and reached the blastocyst stage within 24 h of culturing. This study reports the world's first successful transfer of frozen–thawed Djungarian hamster embryos yielding term pups. Taken together, the results of this study demonstrate the possibility of applying some key reproductive technologies, that is, embryo freezing/cryopreservation and in vitro culture, to Djungarian hamsters.     

Effect of Oviductal Proteins on Structural and Functional Characteristics of Cryopreserved Sperm in Murrah Buffaloes*

The present study was undertaken to elucidate the effect of non‐luteal oviductal proteins on sperm characteristics in Murrah buffaloes. Oviducts from healthy buffaloes were collected immediately after slaughter and the oestrous cycle phase was determined as either luteal or non‐luteal based on ovarian morphology. Non‐luteal oviducts (n = 80) were flushed from the isthmic end of the oviduct with PBS, fluid was centrifuged at 10 000 g at 4°C for 20 min and then dialysed and clarified. The supernatant obtained was lyophilized to concentrate the protein and stored at ?20°C till use. Sixteen good quality ejaculates from four Murrah buffalo bulls were collected using an artificial vagina. After fresh semen analysis, each ejaculate was split into two parts and extended in Tris–citrate–egg yolk glycerol dilutor. Part I of the split ejaculate was treated with non‐luteal oviductal proteins at the dose rate of 1 mg/ml of diluted semen, while part II remained as control. The extended semen was equilibrated for 4 h at 5°C, filled in 0.5 ml French straws, exposed to LN₂ vapour, plunged into LN₂ and then stored at ?196°C. The equilibrated and frozen–thawed semen was evaluated for sperm motility, viability, acrosomal integrity, cervical mucus penetration test and hypo‐osmotic sperm swelling test (HOST). In frozen–thawed semen, the percentage of sperm motility, viability and acrosomal integrity was significantly (p < 0.05) higher in the treatment group compared to the control group. The incorporation of non‐luteal oviductal proteins in the extender increased the ability of sperm to penetrate cervical mucus both after equilibration and the freeze‐thaw process. Similarly, the proportion of sperm with intact plasma membrane, as revealed by HOST values, was also significantly (p < 0.05) higher in the treatment group (32.6%) than the control group (27%) in frozen–thawed semen. It was inferred that incorporation of non‐luteal whole oviductal fluid proteins improved the sperm quality in frozen–thawed semen in Murrah buffaloes.     

In vitro Evaluation of in vivo Fertilizing Ability of Frozen Rabbit Semen

The present work studied different spermatozoa parameters and the ability of frozen rabbit spermatozoa to fertilize, in vitro, in vivo‐matured oocytes, as a test to predict their in vivo fertility and prolificacy. Semen from rabbit bucks was frozen using two freezing protocols [in a freezer at ?30°C or in liquid nitrogen vapour (LNV)]. For the in vivo trial, females were inseminated with frozen‐thawed spermatozoa. Oocytes used for in vitro testing were recovered 14 h after ovulation induction from donors and co‐incubated with 2 × 10⁶ frozen‐thawed spermatozoa during 4 h at 37°C in Tyrode's medium under an atmosphere of 5% CO₂ in air with maximal humidity. After co‐incubation period, presumptive zygotes were cultured in TCM199 supplemented with 20% foetal bovine serum (FBS), under the same conditions described above. Although no statistical differences were observed between freezing protocols in seminal parameters [motility rate: 40 and 35%, VCL: 35 and 46 μm/s, amplitude of lateral head displacement (ALH): 1.7 and 2.4 μm, for semen frozen at ?30°C and in LNV, respectively], significant differences were noted in the fertilizing ability in vivo and in vitro. Semen frozen at ?30°C showed the highest fertilizing ability in vitro (26.7% vs 6.2 and 8.7% for semen frozen at ?30°C, in LNV and fresh semen, respectively) and the lowest fertility rate in vivo (21.7% vs 64.2% and 70.6% for semen frozen at ?30°C, in LNV and fresh semen, respectively). Sperm frozen at ?30°C seemed to be more capacitated.     

Effects of resveratrol treatment on mitochondria and subsequent embryonic development of bovine blastocysts cryopreserved by slow freezing

This study evaluated the effects of cryopreservation by slow freezing on the mitochondrial function, DNA integrity, and developmental ability of bovine embryos and examined whether resveratrol treatment of the frozen‐thawed blastocysts improved embryonic viability. In vitro produced bovine embryos were subjected to slow freezing. After thawing, the ATP content and mitochondrial DNA integrity (mtDNA), determined by real‐time PCR targeting short and long mitochondrial sequences, was found to be lower in frozen‐thawed embryos than in fresh embryos, and mtDNA copy number was significantly reduced during the 24‐hr incubation post warming. Furthermore, immunostaining against double‐strand DNA revealed DNA damage in frozen‐thawed embryos. When frozen‐thawed embryos were incubated in the medium containing 0.5 µM resveratrol, SIRT1 expression, and survival rate of the embryos significantly improved compared with the vehicle‐treated embryos. In addition, cell‐free mtDNA content in medium was higher in case of resveratrol‐treated embryos than of vehicle‐treated embryos. In conclusion, slow freezing affects mitochondrial integrity and function in the blastocysts. In the frozen‐thawed embryos, mitochondria were removed during post‐thawing incubation and resveratrol enhanced the process, resulting in improved survivability of the embryos.     

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.     

Attempt to produce nuclear transferred primordial germ cells using electrofusion in domestic chicken

As a step to develop a somatic nuclear transfer technique for avian species, an attempt to produce somatic nuclear transferred primordial germ cells (PGC) in the domestic chicken was carried out. Primordial germ cells and embryonic blood cells (EBC) were collected from 2‐day‐old embryos and the nuclei were transferred from EBC into PGC by electrofusion. The most efficient pearl chain was developed when a 350‐V/cm AC field was applied for 60 s. Cell fusion between PGC and EBC was most effective when 4‐kV/cm DC pulses, 60 µs pulse width, were applied three times to a cell suspension dispersed in 0.2 or 0.25 mol/L saccharose solution. The present results provide basic information for the production of somatic cell nuclear transferred chickens using PGC as the nuclear recipient.     

Influence of the Preservation Temperature (37, 20, 4, −196°C) and the Mixing of Semen over Sperm Quality of Majorera Bucks

This study assessed the effect of different semen storage temperatures and the influence of semen pooling in semen viability. In experiment 1, semen samples (n = 30) of five Majorera bucks were individually processed [Individual semen (IS)] and after the first dilution (Tris‐yolk extender), semen‐diluted aliquots from each male were pooled semen (PS). Thereafter, semen samples (IS and PS) were preserved as fresh semen (37 and 20°C), chilled semen (4°C) and frozen semen. Sperm motility and the percentage of abnormal sperm cells and intact membrane acrosomes were defined. Semen preservation at 20 and 4°C did not modify the quality of spermatozoa for the first 24 h, but the conservation at 37°C caused a dramatic fall in the semen motility from 12 h onwards. Furthermore, the longevity of frozen‐thawed semen was limited to 4–6 h. No differences were observed in semen parameters when PS was compared with semen from individual males in any of the preservation protocols assessed. In experiment 2, 120 goats were distributed in four experimental groups: in group fresh individual semen (FIS, n = 30) and group frozen‐thawed individual semen (FTIS, n = 30), does were transcervically inseminated with fresh semen and frozen‐thawed semen from each individual male, respectively, and in group fresh pooled semen (FPS, n = 30) and group frozen‐thawed pooled semen (FTPS, n = 30), goats were transcervically inseminated with FPS and FTPS, respectively. The kidding rate was very close in the FIS and FPS groups (70.0% and 73.7%, respectively), and no significant differences were observed in the fertility rate between FTIS and FTPS. The results of this study confirmed that semen samples may be preserved satisfactorily for 24 h both at 20 and 4°C. In addition, the mixture of semen of different bucks did not significantly modify the semen parameters when compared with semen from individual males.     

Induction of superovulation using inhibin antiserum and competence of embryo development in wild large Japanese field mice (Apodemus speciosus)

Seasonally, bred wild mice provide a unique bioresource, with high genetic diversity that differs from wild‐derived mice and laboratory mice. This study aimed to establish an alternative superovulation method using wild large Japanese field mice (Apodemus speciosus) as the model species. Specifically, we investigated how the application of inhibin antiserum and equine chorionic gonadotropin (IASe) during both the reproductive and non‐reproductive seasons impact the ovulation rate and competence of embryo development after in vitro fertilization (IVF) with fresh and cryopreserved sperm. When the wild mice were superovulated by injecting eCG followed by human chorionic gonadotropin (hCG), few oocytes were collected during the reproductive and non‐reproductive seasons. In comparison, the number of ovulated oocytes was dramatically enhanced by the administration of IASe, followed by isolation of ovulated oocytes 24 hr after 30 IU hCG administration. The IVF oocytes that were in vitro cultured (IVC) with medium containing serum further developed to the 2‐ and/or 4‐cell stage using both fresh and frozen‐thawed sperm. In conclusion, we successfully established an alternative protocol for collecting ovulated oocytes from wild large Japanese field mice by administering IASe and hCG during both the reproductive and non‐reproductive seasons. This study is the first to develop IVF–IVC wild large Japanese field mice beyond the 2‐ and/or 4‐cell stage in vitro using fresh and cryopreserved sperm. This approach could be used in other species of wild or endangered mice to reduce the number of animals used for experiments, or in maintaining stocks of germ cells or embryos.     

The effect of cooling to different subzero temperatures on dog sperm cryosurvival

The objective was to assess the effect of cooling to different subzero temperatures around ice formation (?5°C) on dog sperm cryosurvival and plasma membrane fluidity. Semen was centrifuged, and sperm were resuspended in a Tris‐egg yolk medium (3% glycerol). Diluted sperm were cooled from 22 to 5°C, and then, a Tris‐egg yolk medium containing 7% glycerol was added (final concentration of 5% glycerol and 200 × 10⁶ cells/ml). Sperm were packaged in 0.5‐ml plastic straws, and equilibration was done 16 hr at 5°C before freezing. I. Straws (n = 47) at 5°C were exposed to nitrogen vapours to determine the freezing point. II. Other straws (from different ejaculates) processed as mentioned, were further cooled to ?3, ?5 or ?7°C and immediately rewarmed in a water bath at 37°C. Motility, plasma membrane functionality and acrosome integrity were assessed. III. Other straws (from different ejaculates) processed as mentioned were further cooled to ?3 or ?5°C, frozen over nitrogen vapours and stored in liquid nitrogen for one month. Straws were thawed in a water bath at 38°C for 30 s. Motility, plasma membrane functionality, plasma membrane integrity, acrosome integrity, capacitation status and plasma membrane fluidity were assessed. Ice nucleation temperature was ?14.3 ± 2.05°C (mean ± SD); cooling to +5, ?3, ?5 and ?7°C, without freezing, produces no differences on sperm quality between target temperatures; cooling to +5, ?3, and ?5°C produced no differences on sperm survival and plasma membrane fluidity after freeze–thawing. In conclusion, cooling of dog spermatozoa to different subzero temperatures did not improve sperm cryosurvival and had no effect on plasma membrane fluidity after thawing.     

Selection based on morphological features of porcine embryos produced by in vitro fertilization: Timing of early cleavages and the effect of polyspermy

The aim of this study was to examine whether a morphological approach is efficient for selecting high‐quality porcine embryos produced by in vitro fertilization (IVF) under high polyspermy conditions. Frozen‐thawed Meishan epididymal spermatozoa showing moderate and high polyspermy were subjected to IVF (1 × 10⁵ sperms/ml). Under conditions of moderate polyspermy, 4‐cell embryos selected at 48 hr after IVF (single selection) and 8‐cell embryos selected at 79 hr after IVF from the collected 4‐cell embryos (double selection) showed high developmental competence. Likewise, 4‐ and 8‐cell embryos produced by IVF under high polyspermy conditions also showed high competence for development to blastocysts. However, blastocysts derived from high polyspermy conditions had significantly fewer cells than those produced under moderate polyspermy conditions. Furthermore, the frequency of nuclear and chromosomal abnormalities in 4‐ and 8‐cell embryos produced under conditions of high polyspermy was significantly (p < .05) higher in comparison to moderate polyspermy conditions. These findings suggest that although high polyspermy affects the frequency of nuclear and chromosomal anomalies in porcine IVF embryos, subsequent selection based on morphological features of 4‐ and 8‐cell embryos even under high polyspermy conditions, could be an alternative option for selecting porcine IVF embryos with high development ability.     

Testing Usability of Butylated Hydroxytoluene in Conservation of Goat Semen

The objective of this study was to investigate whether butylated hydroxytoluene (BHT) could be used as a suitable supporter or alternative of egg yolk during preservation of goat spermatozoa. Three in vitro experiments and a fertility test were conducted to evaluate the effect of BHT on viability of chilled‐stored semen as well as motility and kidding rate of frozen‐thawed spermatozoa. In the first two experiments, ejaculates (n = 30/experiment) were collected from 10 bucks, split, diluted with egg yolk‐based and egg yolk‐free extenders supplemented with or without 0.3, 0.6, 2, 5 and 8 mm BHT and stored at 5°C for 168 h. In the third experiment, 30 ejaculates were collected from the above‐mentioned bucks, split and diluted with egg yolk‐free extenders supplemented with or without 0.3, 0.6 and 0.9 mm BHT and egg yolk‐based extenders supplemented with or without 5 mm BHT. Diluted semen was cooled to 5°C over a period of 4 h, frozen and thawed in the form of 0.3‐ml pellets. In the fertility test, 75 ejaculates were collected from two proven fertile bucks, split, diluted with egg yolk‐free extenders containing 0.6 mm BHT and egg yolk‐based extenders supplemented with or without 5 mm BHT, frozen and thawed as described above. An insemination volume of 0.6 ml containing 120–140 × 10⁶ progressively motile spermatozoa was used for a single cervical insemination of cloprostenol‐synchronized does (n = 230). The results showed that addition of 5 mm BHT to egg yolk‐deficient (2.5%) extenders significantly improved viability of chilled‐stored semen together with motility (48.5%) and fertility (62.5%) of frozen‐thawed spermatozoa. Replacement of egg yolk in semen extenders by 0.6 mm BHT could sustain not only viability of chilled‐stored semen but also post‐thaw motility (47.5%) and fertility (53.75%) of frozen‐thawed spermatozoa. In conclusion, supplementation of semen diluents with BHT can ameliorate preservability of goat sperm.     

Vitrification for bovine embryos with low‐quality grade

This study was conducted to examine the utility of vitrification for bovine embryos with low‐quality grade, and simple cryoprotectants dilution method for practitioners. In Experiment 1, survival of frozen embryos was compared with that of vitrified embryos using minimum volume cooling (MVC). Then, vitrified embryos were used to confirm the optimum sucrose concentration in Experiment 2. The survival rates of embryos that had been vitrified following diluted cryoprotectants with the one‐step in‐straw method were compared with those of fresh control embryos in Experiment 3. Frozen‐thawed or vitrified‐warmed blastocysts were cultured with TCM‐199 supplemented with 100 μmol/L beta‐mercaptoethanol +5% fetal bovine serum at 38.5°C in an atmosphere of 5% CO₂ in air, their survival after 24 hr were compared. The development to term of fair quality in vivo embryos after vitrification was examined in Experiment 4. Results show that survival rates of frozen‐thawed embryos were lower (p < .05) than that of vitrified‐warmed ones. When vitrified embryos were warmed in 0.3 mol/L sucrose in straws, their survival rate was 100%. The total cell numbers of vitrified‐warmed embryos were comparable to those of fresh control embryos. The six calves from 13 vitrified embryos were delivered in Experiment 4. These results indicate that MVC vitrification following one‐step cryoprotectants dilution is utilized to preserve low‐quality bovine embryos.     

B cell differentiation in the bursa of Fabricius and spleen of embryos and chicks immediately after hatching

Flow cytometric analysis and immunohistochemical observation were used to qualitatively and quantitatively clarify the nature of B cell differentiation in the bursa of Fabricius of chick embryos and to determine the timing of antibody class switching in chicken spleens based on positivity of IgM and IgG on and in the cells. In the bursa, the sIgM‐positive cell population formed from the 12^th to 15^th day of embryogenesis. The proportion of sIgM‐high expressing (sIgM^high) cells was lower among bursacytes than splenocytes of hatched chicks, suggesting that the sIgM^high bursacytes are to be released to peripheral sites. The proportion of sIgM^high cells was higher at 0 days old than at any other examined stage of development. Colonization of the spleen by B cells occurred between the 18^th day of embryogenesis and 0 days old. Antibody class switching was thought to start in the spleen between 1 and 2 weeks of age, because IgG‐positive cells were present in the spleen of 2‐week‐old chicks, but not 0‐day‐old or 1‐week‐old chicks.     

Effects of Incubation Temperature and Semen Pooling on the Viability of Fresh,Chilled and Freeze‐Thawed Canine Semen Samples

This study assessed the effects of different incubation temperatures on semen viability and the influence of pooling on semen longevity. In experiment 1, semen samples were collected from five dogs, individually processed (individual semen: IS) and then aliquots from each male were pooled (pooled semen: PS). Semen samples (IS and PS) were diluted in a Tris‐glucose‐yolk extender and preserved as fresh (37 and 25°C) and chilled semen (4°C). Sperm motility and the percentages of sperm abnormalities and acrosome membrane integrity were assessed for 24 h. Storage at 25 or 4°C for the first 24 h yielded similar semen quality, but incubation at 37°C caused drastic reduction in sperm motility from 8 h of incubation onwards. In experiment 2, the semen was processed in the same way to that of experiment 1 and then preserved at 25 or 4°C until semen inactivation. Semen that was incubated at 25°C became completely inactive after 3–4 days of storage, while semen that was preserved at 4°C presented with more gradually decreased sperm motility (mean values of 40–60% for the first 8 days). In addition, the mixing of semen was only observed to influence the sperm quality of the samples stored at 4°C. In experiment 3, semen was collected from five dogs, pooled and frozen in liquid nitrogen; after thawing, it was preserved at 37, 25, 15 and 4°C, and the sperm quality was defined. The motility of the freeze‐thawed semen samples decreased quickly in the first 4 h after thawing, regardless of the preservation temperature of the thawed semen. This study confirmed that semen preserved at 37°C should be used within a maximum of 12 h, while the semen stored at 25°C shows acceptable quality for 24 h. Chilled semen presented highest most sustainable quality, especially when semen is processed as pooled semen.     

The effects of early‐age thermal manipulation and daily short‐term fasting on performance and body temperatures in broiler exposed to heat stress

This study was conducted to investigate the effects of thermal manipulation at 5 days of age and short‐term fasting during the warmest part of the day on responses to prolonged heat stress of broilers. A total of 240‐day‐old Ross 308 female broiler chicks were divided into three groups: control, thermal manipulation (chicks were exposed to 36 °C for 24 h at 5 days of age) and short‐term fasting during the warmest part of the day (10.00–17.00 h). Prolonged heat stress was induced daily from 28 to 42 days by heating until the ambient temperature reached 32–35 °C between 10.00 and 17.00 h. Both thermal manipulation and short‐term fasting resulted in a decrease in rectal temperatures and haematocrit values at 35 and 41 days of age. Thermal manipulation improved body weight, feed consumption and feed conversion. However, short‐term fasting caused a reduction in body weight and a deterioration in feed conversion. Short‐term fasting lowered the percentages of carcass, whereas thermal manipulation highered breast yield. Both thermal manipulation and short‐term fasting decreased heart mass and abdominal fat.