Pregnancy loss in heifers after artificial insemination with frozen-thawed,sex-sorted,re-frozen-thawed dairy bull sperm

The use of sex-sorted sperm by the dairy industry is often limited by the geographical distance between potential sires and the sex-sorting facility. One method that may be used to overcome this limitation is sex-sorting sperm that have been previously frozen, or transported to the sorting facility as cooled liquid semen. In this study the in vivo fertility of frozen-thawed, sex-sorted, re-frozen-thawed (FSF) and cooled, sex-sorted, frozen-thawed (CSF) bull sperm was determined after artificial insemination (AI) of Holstein heifers. Semen from two bulls was frozen in straws, or transported to the sorting facility in an egg yolk diluent at 5 °C over 24 h. Thawed or re-warmed semen was processed through a PureSperm^® density gradient, and sperm were sorted for sex and frozen (2 or 4 × 10⁶ sperm/straw). Synchronised heifers (n = 183) were inseminated with either non-sorted control sperm (Control; 20 × 10⁶ dose) or with FSF or CSF ‘X’ sperm (2 or 4 × 10⁶/dose). Pregnancy rates (detected at 7–9 weeks) after AI with control sperm were higher than with FSF or CSF sperm (57.4 vs. 4.1 and 7.3% respectively; p < 0.001). There was a significant difference between bulls (Bull 1: Control 63.0%, FSF 8.6%, CSF 10.0%; Bull 2: Control 45.5%, FSF 0%, CSF 4.8%; p = 0.001). Five out of six (83.3%) pregnancies produced with sexed sperm were lost after pregnancy diagnosis. The exception was one heifer inseminated with CSF sperm (2 million sperm dose), which produced a heifer calf. In the non-sorted control group, three pregnancies were lost (8.3%) and three stillbirths occurred (8.3%). The low fertility and high rate of pregnancy loss in the sexed groups, in addition to environmental influences, may be attributed to impaired sperm function caused by sex-sorting and re-freezing, leading to poor embryo quality or altered gene expression. More precise timing of insemination and higher sperm doses might improve the fertility of FSF sperm. Moreover, the in vitro function of double-frozen sexed compared with non-sorted sperm requires further investigation.     

Embryo production after in vitro fertilization with frozen-thawed, sex-sorted, re-frozen-thawed bull sperm

The objective of this study was to determine the in vitro fertilizing capacity of bull sperm derived from fresh or frozen samples and subjected to sex sorting and re-cryopreservation. Four sperm types were assessed for their ability to fertilize and sustain early embryo development in vitro. Semen from three Bos taurus bulls of different breeds (Jersey, Holstein and Simmental) was collected and either sorted immediately and then frozen (SF) or frozen for later sorting. Frozen sperm destined for sorting were thawed, sex-sorted, and re-frozen (FSF) or thawed, sex-sorted (FS), and used immediately for in vitro fertilization (IVF). Frozen-thawed nonsorted semen from the same ejaculate was used as a control. Oocytes from donor cows were aspirated via ovum pick-up and matured in vitro prior to IVF and culture. On average, 19.0 ± 1.7 (mean ± SEM) oocytes were aspirated per donor cow, of which 74.4 ± 2.2% were selected for maturation. The proportion of cleaved embryos (Day 3) did not differ between sperm groups (P = 0.91). Likewise, IVF with FSF sperm resulted in similar Day 7 blastocyst rates (as a percentage of total oocytes) as those of control, SF, and FS sperm (FSF, 34.5 ± 4.7; control, 32.2 ± 4.6; SF, 35.9 ± 4.8; and FS, 26.9 ± 4.1%; P = 0.23). These encouraging results show that frozen-thawed sex-sorted sperm may be re-frozen and used for in vitro embryo production with similar blastocyst production as that of nonsorted frozen-thawed and sex-sorted frozen-thawed sperm.     

Dietary omega-3 fatty acids (fish oils) have limited effects on boar semen stored at 17 °C or cryopreserved

To evaluate the influence of dietary supplementation of omega-3 polyunsaturated fatty acids (n-3 PUFA) on storage of boar semen, three experiments were conducted: two involved long-term, fresh semen storage (Exp. 1 and Exp. 2), whereas the other involved cryopreservation (Exp. 3). Boars were allocated randomly to three dietary treatments (for 6-7 mo). In addition to a daily allowance of 2.5 kg of a basal diet, they received: 1) 62 g of hydrogenated animal fat (AF); 2) 60 g of menhaden oil (MO), containing 18% docosahexanoic acid (DHA) and 15% eicosapentanoic acid (EPA); or 3) 60 g of tuna oil (TO), containing 33% DHA and 6.5% EPA. In Experiment 1 (n = 26) and Experiment 2 (n = 18), semen was cooled and stored in vitro for several days at 17 °C before assessment, whereas in Experiment 3 (n = 18), viability, motility, acrosomal integrity, susceptibility to peroxidation (LPO), and DNA fragmentation were determined in fresh and frozen-thawed sperm. In Experiment 1, sperm from boars fed TO had better resistance to fresh storage; even after 7 or 9 d of storage at 17 °C, there were more (P = 0.03) motile sperm in boars fed TO (>60%) than in those fed AF or MO. In Experiment 2, fish oil supplementation did not influence any aspect of sperm quality during semen storage (P > 0.10). In Experiment 3, cryopreservation decreased the proportion of motile and viable frozen-thawed sperm as well as acrosomal integrity and increased DNA fragmentation and LPO (P < 0.01) relative to fresh semen, although sperm quality was unaffected by treatments (P > 0.09). In conclusion, although adding fish oil to the diet failed to significantly improve the quality of cryopreserved boar sperm, inconsistent responses of long-term storage of cooled sperm to dietary n-3 PUFA supplementation warrant further investigation.     

Zwitterionic buffers preserve ram semen quality more efficiently than TRIS during storage at 15 °C

This study was designed to evaluate the effect of various buffers on the storage of ram semen at 15 °C. Second ejaculates from six adult males were collected using an artificial vagina and diluted in either MOPS, TRIS, TES, HEPES, citrate, or phosphate-based extenders. Semen samples were stored at 15 °C and the sperm motility and viability (membrane integrity) variables assessed after 0, 24 and 48 h intervals. Significantly higher progressive sperm motility rates were recorded at 0 h of storage, and higher motile and progressive sperm motility at 24 and 48 h, when zwitterionic-based extenders (MOPS, TES and HEPES) were used, compared to citrate, TRIS, and phosphate-based extenders—with the last group showing a drastic reduction in sperm motility during storage. The zwitterionic groups were also superior to the other treatments in terms of sperm velocity (straight line velocity, VSL; curvilinear velocity, VCL; average path velocity, VAP) at 0 h of storage, although at 24 and 48 h the differences were minimal in the CITRATE group—regarding all velocity variables, and in the TRIS group, regarding the VCL parameter. Sperm diluted in the TRIS-based extender showed a marked increase in the proportion of circular sperm trajectories (lower sperm linearity, LIN, and straightness, STR), and a decrease in the VAP. The reduction in the vigour of the sperm in the TRIS extender (measured by VCL) was less pronounced than in the other groups. At the same time, VSL was reduced, as more sperm moved in circles, and the amplitude of lateral head displacement (ALH) was also dramatically increased. The CITRATE diluent recorded intermediate results—between that of TRIS and the other treatment groups, regarding the variables related to the quality of sperm movement at 0 h storage. However, following CITRATE dilution, semen underwent a clear improvement after a period of 24 and 48 h, so that differences with the zwitterionic groups were attenuated or disappeared. Similarly, the CITRATE group obtained similar or higher viable sperm values, compared to zwitterionic buffers during storage. The TRIS, and particularly the PHOSPHATE diluents, recorded poorer sperm viability after 24 and 48 h of storage. It was concluded that zwitterion-based buffers may be an acceptable alternative for inclusion in the composition of diluents for chilled ram semen storage. On the other hand, TRIS may be seen as having caused drastic modifications of certain sperm kinematic parameters during storage at 15 °C.     

Changes in subpopulations of boar sperm defined according to viability and plasma and acrosome membrane status observed during storage at 15 degrees C

Four boar ejaculates were preserved for 42 d at 15 °C to examine the changes produced in the quality of sperm membranes according to their response to a combined short hypoosmotic swelling test (sHOST) plus viability test designated the sHV test. Every 1 or 2 d, a sample from each ejaculate preserved in long-term extender was subjected to sperm motility determination and the sHV test. Through simultaneous examination by phase contrast and fluorescence microscopy, three subpopulations of sperm were identified according to their response to sHOST challenge and their viability status. In the subpopulations scoring positive in the sHOST, a further four sperm subpopulations were defined according to their viability and acrosome status. All the sperm subpopulations differed in terms of changes in their proportions produced during the course of preservation and individual differences among ejaculates were detected in terms of relationships shown among subpopulations. The combined sHOST/viability test was able to identify sperm subpopulations with the strongest plasma and acrosome membranes as well as a subpopulation of sperm that had undergone a true acrosome reaction.     

Microencapsulation of canine sperm and its preservation at 4 °C

The objective of this study was to develop a preservation method for canine sperm using microencapsulation. Pooled ejaculates from three beagles (Canis familiaris) were extended in egg yolk Tris extender and were encapsulated in gel (alginate only) or polycation (poly-l-lysine membrane bound) microcapsules at 0.75% and 1.0% alginate concentration. In Experiment 1, characteristics of microcapsule and microencapsulated sperm were evaluated during chilling storage for 48 h. Gel microcapsules at 0.75% alginate concentration had a teardrop-like structure with fragility, whereas those at 1.0% alginate had a solid spherical structure. In all groups, diameter of the microcapsules increased with duration of storage (P < 0.05). Alginate concentration did not affect the sperm recovery rate from microcapsules. Total average recovery rate of sperm from polycation microcapsules was lower than that of gel microcapsules (P < 0.05). Progressive motility of polycation microencapsulated sperm and unencapsulated sperm (control) was higher than that of the gel microencapsulated sperm, both at 0.75% and 1.0% alginate concentration (P < 0.05), although viability of sperm was similar among the three groups. In Experiment 2, to evaluate the sperm longevity after chilling storage, sperm were microencapsulated in polycation microcapsules at 1.0% alginate concentration, stored at 4 °C for 0, 1, 4, and 7 d, and then cultured at 38.5 °C for 0, 6, and 24 h. Progressive motility and viability of microencapsulated sperm were higher than those of unencapsulated spermatozoa at 0 to 24 h of culture after 4 and 7 d of chilling storage (P < 0.05). In conclusion, polycation microencapsulation at 1.0% alginate concentration can be successfully applied for chilling storage of canine sperm by maintaining motility and viability for up to 7 d.     

Acrosomal integrity, viability, and DNA damage of sperm from dasyurid marsupials after freezing or freeze drying

Dasyurids are a diverse group of Australian native carnivores and insectivores that contains several threatened species. Despite successful cryopreservation of sperm from several marsupials, only 3% postthaw motility is reported for dasyurid marsupials. This study examined sperm preservation in the fat-tailed dunnart (Sminthopsis crassicaudata), an experimental model, with supplementary observations on the eastern quoll (Dasyurus viverrinus) and northern quoll (Dasyurus hallucatus). In S. crassicaudata, a toxicity trial demonstrated that incubation with up to 40% glycerol did not reduce sperm viability, suggesting that glycerol is not toxic to dasyurids. On the basis of this finding, S. crassicaudata, D. viverrinus, and D. hallucatus sperm were extended to a final concentration of 20% or 40% glycerol in Tris-citrate fructose and frozen in liquid nitrogen vapor. Postthaw sperm from all three species were nonmotile, and vital staining (SYBR14 and propidium iodide) indicated that sperm were nonviable. However, there was no evidence suggesting disruption of normal gross morphology or loss of acrosomal integrity when assessed by Bryan's staining. After freeze drying, Bryan's staining indicated that approximately 80% of S. crassicaudata sperm had normal acrosomes and no head loss. Despite being nonviable, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling showed that S. crassicaudata sperm frozen in 40% glycerol or freeze-dried had no DNA damage compared with fresh controls. This study has described a method for preservation of the dasyurid sperm nuclei, but continued studies are required to achieve viable motile sperm and establish tools for the long-term storage of dasyurid sperm.     

Effect of blood serum, caffeine and heparin on in vitro phagocytosis of frozen-thawed bull sperm by neutrophils derived from the peripheral blood of cows

Although polymorphonuclear leukocytes (PMNs) are recruited into the uterine lumen to phagocytize sperm, factors controlling the phagocytotic ability of PMNs in cattle are not well documented. The objective was to determine the effects of blood serum, caffeine, and heparin on chemotaxis of PMNs for sperm and phagocytosis of sperm by PMNs in cows. Polymorphonuclear leukocytes were obtained (centrifugation) from a cow's peripheral blood. In Experiment 1, the chemotactic activity of PMNs increased (P < 0.01) when fresh serum was included in the medium (1226 cells/mm² in serum vs. 1110 cells/mm² in BSA), regardless of the presence of sperm, whereas heat-inactivated serum (1099 cells/mm²) did not affect their activity (P = 0.65). Phagocytosis of live and dead sperm by PMNs both increased (P < 0.01) in the presence of fresh serum (incidences of 54.5 and 48.0%, respectively), but stimulation was decreased (P < 0.01) by supplementation of the medium with ≥1 mM caffeine (20.6-30.3%). Serum-stimulated chemotactic activity of PMNs (1218 cells/mm²) was also decreased (P < 0.01) in the presence of caffeine (1090 cells/mm²). Furthermore, supplementation of PMNs with heparin in the presence of serum decreased (P < 0.01) both phagocytotic (from 43.8% to 21.5-31.7%) and chemotactic activities of PMNs (from 1124 to 1048-1108 cells/mm²). We inferred that opsonization in the presence of active complement stimulated phagocytotic and chemotactic activities of PMNs, and that both caffeine and heparin decreased serum-stimulated phagocytotic and chemotactic activities of PMNs.     

Reducing the time rabbit sperm are held at 5 °C negatively affects their fertilizing ability after cryopreservation

Cooling sperm to and equilibrating the sperm at 5 °C require the most time in any sperm cryopreservation protocol. Reducing the time required for these phases would simplify sperm freezing protocols and allow greater number of ejaculates to be processed and frozen in a given time. This study determined how holding rabbit sperm at 5 °C for different lengths of time (0, 10, 15, 20, 30, or 45 minutes) affected the quality of rabbit sperm, measured by in vitro assays, and if reducing the cooling time to only 10 minutes affected the fertilizing ability of the sperm. Reducing the time sperm were held at 5 °C to 10 minutes did not affect the in vitro quality of the sperm (percent motile and with intact plasma membranes), although eliminating the cooling phase completely (directly freezing the sperm from room temperature) decreased in vitro assessed sperm quality (P < 0.01). However, reducing the time sperm were held at 5 °C, from 45 to 10 minutes, negatively affected the fertilizing ability of sperm in vivo (P < 0.05). In conclusion, completely eliminating cooling rabbit sperm to 5 °C before freezing is detrimental for rabbit sperm cryosurvival, and although shortening the time sperm are held at 5 °C to 10 minutes does not reduce in vitro sperm quality, it does reduce the fertility of rabbit sperm. Therefore, the length of time rabbit sperm equilibrate at 5 °C is crucial to the fertilizing ability of rabbit sperm and must be longer than 10 minutes. Currently, it is not known if holding rabbit sperm at 5 °C for less than 45 minutes will affect sperm fertilizing ability.     

Effect of glycerol concentration, Equex STM® supplementation and liquid storage prior to freezing on the motility and acrosome integrity of frozen-thawed epididymal alpaca (Vicugna pacos) sperm

Two experiments were conducted to determine the effects of glycerol concentration and Equex STM® paste on the post-thaw motility and acrosome integrity of epididymal alpaca sperm. In Experiment 1, epididymal sperm were harvested from male alpacas, diluted, and cooled to 4 °C in a Lactose cooling extender, and pellet-frozen in a Lactose cryodiluent containing final glycerol concentrations of 2, 3, or 4%. In Experiment 2, epididymal sperm were diluted in Biladyl®, cooled to 4 °C, stored at that temperature for 18-24 h, and further diluted with Biladyl® without or with Equex STM® paste (final concentration 1% v:v) before pellet freezing. In Experiment 1, sperm motility was not affected by glycerol concentration immediately (2%: 16.1 ± 4.6%; 3%: 20.5 ± 5.9% and 4%: 18.5 ± 6.6%; P > 0.05) or 3h post thaw (< 5% for all groups; P > 0.05). Post-thaw acrosome integrity was similar for sperm frozen in 2% (83.6 ± 1.6%), 3% (81.3 ± 2.0%) and 4% glycerol (84.8 ± 2.0%; P > 0.05) but was higher 3h post-thaw for sperm frozen in 3% (75.7 ± 3.8%) and 4% (77.2 ± 4.1%) than 2% glycerol (66.9 ± 2.7%; P < 0.05). In Experiment 2, sperm motility was higher immediately after thawing for sperm frozen in the presence of Equex STM® (Equex®: 21.5 ± 3.5%; control: 14.4 ± 2.1%; P < 0.05) but was similar at 3h post-thaw (P > 0.05). Acrosome integrity was similar for sperm frozen with or without Equex STM® paste immediately (control: 89.6 ± 1.2%; Equex®: 91.1 ± 1.4%; P > 0.05) and 3 h post-thaw (control: 69.3 ± 3.7%; Equex®: 59.9 ± 5.8%; P > 0.05). Sperm cryopreserved in medium containing 3-4% glycerol and 1% Equex STM® retained the best motility and acrosome integrity, even after liquid storage for 18-24 h at 4 °C prior to cryopreservation.     

Effect of storage of domestic cat (Felis catus) epididymides at 5 °C on sperm quality and cryopreservation

Postmortem sperm recovery from the epididymides may constitute a powerful tool for the conservation of valuable genetic material. The domestic cat (Felis catus) is a good model for wild felids and, using this model, we have explored the effect of epididymides storage time on sperm motility and percentage of intact acrosomes upon sperm recovery and after cryopreservation. We also examined the effect of time of sperm equilibration with glycerol before freezing on sperm motility and the percentage of intact acrosomes. Motility varied between sperm recovered from epididymides that were stored for different times. Significant differences were seen in the sperm motility index (SMI) before freezing (55.91 ± 2.02, 48.21 ± 1.47, and 43.03 ± 1.32) and after thawing (51.81 ± 3.02, 41.90 ± 2.14, and 42.35 ± 1.95) of sperm recovered from epididymides stored for 0, 48, or 72 h, respectively. The percentage of intact acrosomes did not vary significantly with storage time (average 60.33 ± 1.38% before and 52.50 ± 1.91% after freezing, respectively). The percentage of normal sperm after different storage times did not differ (average 19.22 ± 1.25% normal sperm after recovery). When epididymides were stored for 72 h, time of sperm equilibration with glycerol (30 vs. 120 min) resulted in significant differences in both motility (SMI = 39.17 ± 2.76 and 45.00 ± 2.65, respectively) and the percentage of intact acrosomes (45.76 ± 4.91% and 60.67 ± 3.64%, respectively) after thawing. In conclusion, best results are achieved when sperm are recovered from epididymides within 24 h of cool storage and when they are equilibrated with glycerol during 120 min before freezing. The current results should be useful in the further development of techniques for the rescue and cryostorage of epididymal spermatozoa of endangered felids.     

Fertilization of C57BL/6 mouse sperm collected from cauda epididymides after preservation or transportation at 4 °C using laser-microdissected oocytes

The C57BL/6 mouse is commonly used to produce transgenic and knockout strains for biomedical research. However, the motility and fertility of its sperm decrease markedly with freezing. Short-term preservation of sperm without freezing can avoid this. Furthermore, such samples can be transported safety without the special skills or equipment needed for the transportation of live animals or frozen products. We evaluated the motility and fertility of sperm collected from cauda epididymides after preservation or transportation at 4 °C. Oocytes with the zona pellucida subjected to laser-microdissection were used to assist fertilization in vitro. Although the motility of sperm gradually decreased with storage (P < 0.05), no disruption of the sperm plasma membrane was seen. The proportion of zona-intact oocytes fertilized with sperm preserved for 0, 24, 48 and 72 h were 70, 14, 5 and 1%, respectively. On the other hand, 45, 20 and 14% of laser-microdissected oocytes were fertilized by sperm preserved for 24, 48 and 72 h, respectively (P < 0.05). The fertility of sperm collected from cauda epididymides of two transgenic strains after transportation at 4 °C were also significantly increased using laser-microdissected oocytes rather than zona-intact oocytes (57 and 68% vs. 5%, P < 0.05). Efficient production of offspring from sperm preserved or transported at 4 °C was achieved using laser-microdissected oocytes. Thus the fertility of sperm preserved or transported at 4 °C could be maintained, although motility gradually decreased with storage. Laser-microdissected oocytes will contribute to the efficient production of embryos and offspring using such preserved sperm samples.     

Scaling of oxygen consumption of Lake Magadi tilapia, a fish living at 37°C

Rates of oxygen consumption were measured in the geothermal, hot spring fish, Oreochromis alcalicus grahami by stopped flow respirometry. At 37° C, routine oxygen consumption followed the allometric relationship: V o₂=0.738 M ^0.75, where V o₂ is ml O₂ h ⁻¹ and M is body mass (g). This represents a routine metabolic rate for a 10 g fish at 37° C of 0.415 ml O₂ g⁻¹ h ⁻¹ (16.4 μmol O₂ g ⁻¹ h ⁻¹). Acutely increasing the temperature from 37 to 42° C significantly elevated the rate of O₂ consumption from 0.739 to 0.970 ml O₂ g ⁻¹ h ⁻¹ ( Q ₁₀=l.72). In the field, O. a. grahami was observed to be 'gulping' air from the surface of the water especially in hot springs that exceeded 40° C. O. a. grahami may utilize aerial respiration when O₂ requirements are high.     

The microbial flora of herring fillets after storage in carbon dioxide, nitrogen or air at 2°C

Herring fillets from the Baltic Sea were stored in glass vessels in air, nitrogen or carbon dioxide (CO₂) at 2°C and the microbial development was studied. The microbiological shelf-life of the herring (the time to reach 10⁷ organisms/g) was prolonged by a factor of 3.5 in CO₂ as compared to air. The corresponding factor in nitrogen was 1.5. The microflora of fresh and spoiled herring was classified. The initial microflora was dominated by coryneforms, Flavobacterium spp., Moraxella -like organisms and Pseudomonas spp. The spoilage-flora in air (after 9 d) was dominated by Pseudomonas spp. and Moraxella -like organisms, and in nitrogen (14 d) Enterobacteriaceae, Vibrionaceae and Lactobacillus spp. were dominant. Homofermentative Lactobacillus spp. were the only organisms isolated from fillets stored in CO₂ (28 d). It was concluded that storing fresh fish in pure CO₂ at low refrigeration temperatures is a method with industrial potential. The method (1) improves the microbiological stability and (2) reduces the microbiological health hazards of the fish.     

In vitro study of the biological activity of RNAs after incubation of hog liver, heart and brain tissue at room temperature

The biological activity of RNA, isolated from tissue which was incubated for 1, 3, or 6 hours at room temperature (simulation of post-mortem conditions), was preserved. However, the different organs used differ from each other. When liver is used, qualitative differences in the in vitro translation products are observed, after one hour incubation at room temperature, whereas when heart and brain are used these differences are not observed. We have also shown that relatively small amounts of post-mortem tissue is sufficient for RNA extraction. When using frozen tissue it is absolutely necessary to add RNase inhibitors during thawing to reduce the loss of biological activity.