Dimethylformamide Improves the In vitro Characteristics of Thawed Stallion Spermatozoa Reducing Sublethal Damage

A total of 42 ejaculates were used in the experiment; six ejaculates per stallion, obtained from seven Pure Spanish stallions (PRE), were split and frozen in freezing media with different concentrations and combinations of cryoprotectant (CPA): (i) Cáceres (skim milk based extender) containing 2.5% glycerol (2.5GL), (ii) Cáceres containing 1.5% glycerol and 1.5% dimethylformamide (1.5%GL–1.5%DMFA), (iii) Cáceres extender supplemented with 1.5% glycerol and 2.5% dimethylformamide (1.5%GL–2.5%DMFA) and (iv) Cáceres extender supplemented with 4% dimethylformamide (4%DMFA). After at least 4 weeks of storage in liquid nitrogen (LN), straws were thawed and semen analysed by computer‐assisted sperm analysis and flow cytometry (membrane lipid architecture (Merocyanine 540), integrity and sublethal damage (YoPro‐1) and mitochondrial membrane potential (JC‐1)). After thawing, better results were observed in samples frozen in 4%DMFA or in combinations of 1.5%GL–2.5%DMFA, in fact total motility increased by 16% in the 4%DMFA group compared to 2.5%GL (P < 0.05). Also, there was an increment in the percentage of progressive motile sperm in the 1.5%GL–2.5%DMFA group (9.8% 2.5GL vs 19% in the 1.5%GL–2.5%DMFA group p < 0.05); also, samples frozen in the 4%DMFA group had more intact (YoPro‐1 negative) sperm post‐thawing, 29.3% in 2.5%GL vs 36.7% in 4%DMFA group (p < 0.05). Membrane lipid architecture was not affected by any of the cryoprotectants tested, while samples frozen in 4%DFMA had a lower percentage of mitochondria with lower membrane potential. It is concluded that DMFA improves the outcome of cryopreservation of stallion spermatozoa mainly reducing sublethal cryodamage.     

Single layer centrifugation of fresh dromedary camel semen improves sperm quality and in vitro fertilization capacity compared with simple sperm washing

Single layer centrifugation (SLC) through a colloid is a tool for selecting viable mammalian spermatozoa but has not been used previously for fresh dromedary camel sperm. Semen from six camels (2 ejaculates/male) was diluted 1:5 (v:v) or 1:10 (v:v) in a Tris–citrate–fructose buffer for mechanical liquefaction by gentle pipetting. Following liquefaction, semen was processed either by SLC or by centrifugation without a colloid (control). Total and progressive motilities, CASA kinematics, vitality and acrosome integrity (eosin–nigrosin) and plasma membrane integrity (Hypo‐osmotic swelling test; HOST), and fertilizing ability in a heterologous assay (zona‐free goat oocytes) were evaluated. Both total (p = .003) and progressive motilities (p = .003) were higher in SLC‐processed than in control semen samples, irrespective of dilution. Positive HOST values increased when using colloid in 1:5 (p = .001) and 1:10 dilution (p = .010). Colloid‐selected sperm had higher penetration rates than controls (p < .001 and p = .02 for 1:5 and 1:10 dilutions, respectively). However, only the SLC sperm at 1:5 dilution showed higher percentages of pronuclear formation (p = .02) than controls. Dilution effect was only significant for total motility before in vitro fertilization, with higher values for the 1:5 dilution (p = .033). The recovery rates of motile sperm between dilutions were similar (26.1% vs 35.4%; p = .226). In conclusion, SLC is a promising tool for selecting functional dromedary camel sperm and warrants more research.     

Effects of Serum Deprivation and Cycloheximide on Cell Cycle of Low and High Passage Porcine Fetal Fibroblasts

Arrest of cells in G0/G1 cell cycle phase is desired for nuclear transfer procedures. Serum starvation and cell cycle inhibitors are different ways to induce synchronization of the cell cycle. This study investigated the effects of serum starvation and cycloheximide (CHX) on the cell cycle of low (5th) and high (15th) passages fetal porcine fibroblasts. Cell cycle phases were determined using fluorescent activated cell sorting. Fifth passage fibroblast cultures had higher (p < 0.05) proportion of cells in G0/G1 only after 72 h of serum starvation (77.60 ± 0.65) when compared with non‐starved cells (71.44 ± 1.88). Serum starvation for all periods tested induced an increase (p < 0.05) on proportion of cells in G0/G1 on the 15th passage. No significant differences were observed on the 5th passage cultures exposed to CHX, although, on the 15th passage an increase on proportion of cells was observed after all periods of exposure (p < 0.05). These data indicates that high passage cells in vitro are more susceptible to serum starvation and CHX G0/G1 synchronization.     

Spatial Response of Mammals to Late Quaternary Environmental Fluctuations

Analyses of fossil mammal faunas from 2945 localities in the United States demonstrate that the geographic ranges of individual species shifted at different times, in different directions, and at different rates in response to late Quaternary environmental fluctuations. The geographic pattern of faunal provinces was similar for the late Pleistocene and late Holocene, but differing environmental gradients resulted in dissimilar species composition for these biogeographic regions. Modern community patterns emerged only in the last few thousand years, and many late Pleistocene communities do not have modern analogs. Faunal heterogeneity was greater in the late Pleistocene.     

Membrane Lipids of the Stallion Spermatozoon in Relation to Sperm Quality and Susceptibility to Lipid Peroxidation

Lipids were extracted from ejaculated spermatozoa from seven individual stallions to distinguish neutral lipids (NL) and polar lipids (PL) and determine their variation among stallions and their relationship with sperm quality and sperm susceptibility to lipid peroxidation. The isolated fatty acids were correlated with sperm quality (membrane integrity, mitochondrial membrane potential (ΔΨm) and expression of active caspases) and the sensitivity of the sperm plasma membrane to LPO. The miristic (C14: 0), palmitic (C16: 0), stearic (C18: 0) and oleic (C18: 1n9) acids were predominant among the NLs. Within the phospholipid fraction, the docosapentanoic acid (C22: 5n6) was dominant, albeit varying among stallions. Surprisingly, the percentage of polyunsaturated fatty acids was positively correlated with sperm quality and a low propensity for LPO, probably because these particular fatty acids provide a higher fluidity of the plasma membrane. The stallion showing the poorest sperm membrane integrity plus a high level of LPO in his ejaculate had a lower percentage (p < 0.05) of this fatty acid in his sperm plasma membranes.