Comparison of Synthetic Oviductal Fluid and G1/G2 Medium under Low‐1 Oxygen Atmosphere on Embryo Production and Pregnancy Rates in Nelore (Bos indicus) Cattle

In this work, we evaluated whether embryo development and pregnancy rates would be affected by culturing bovine Bos indicus embryos in Synthetic Oviductal Fluid with amino acids (SOFaa) or G1/G2 sequential medium under a low‐oxygen atmosphere. Using Ovum Pick Up, we obtained 1,538 oocytes, divided into G1/G2 (n = 783) and SOFaa (n = 755). No difference was observed for blastocyst development among the groups (27.8% ± 14.6 and 34.9% ± 20.0 for G1/G2 and SOFaa respectively, p > 0.05). Transferring the embryos (n = 450) from both groups to recipients resulted in similar pregnancy rates for the G1/G2 (38.4% n = 78/203) compared to the SOFaa (39.7% n = 98/247). Our findings confirm that Bos indicus embryos cultured in SOFaa and G1/G2 under low‐oxygen atmosphere have similar in vitro (blastocyst rate) and in vivo (pregnancy rate) developmental capacity. However, embryos cultured in G1/G2 medium have higher cleavage than those cultured in SOFaa medium.     

Simultaneous steroids measurement in dogs with hyperadrenocorticism using a column-switching liquid chromatography-tandem mass spectrometry method

We developed an analytical method using an on-line column-switching liquid chromatography with triple quadrupole mass spectrometry (LC/MS/MS) for quantifying multiple steroids in serum. Using the developed method, we evaluated the serum concentration of nine steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, 21-deoxycortisol, deoxycorticosterone, progesterone, 17α-OH-progesterone and aldosterone) in dogs with hyperadrenocorticism (HAC). Serum was mixed with stable isotope internal standards and thereafter purified by the automated column-switching system. The limit of detection ranged 2–16 pg/ml for nine steroids. In the baseline samples, five steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, and 17α-OH-progesterone) were detected in all dogs. The concentrations of cortisone, 11-deoxycortisol, and 17α-OH-progesterone in dogs with HAC (n=19) were significantly higher those in dogs without HAC (n=15, P<0.02). After the adrenocorticotropic hormone stimulation test, six steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, 17α-OH-progesterone, and deoxycorticosterone) were above the limit of quantification in all dogs. Cortisol, corticosterone, cortisone, and deoxycorticosterone concentrations of dogs with HAC were significantly higher than those of dogs without HAC (P<0.02). In addition, 11-deoxycortisol and 17α-OH-progesterone concentration was higher in dogs with HAC than in dogs without HAC (P=0.044 and P=0.048, respectively). The on-line column-switching LC/MS/MS would be feasible for measuring multiple steroids in dog serum. The results suggest that cortisone, 11-deoxycortisol, and 17α-OH-progesterone would be related to HAC. Further studies are warranted to assess the clinical feasibility of steroid profile in dogs with HAC.     

Response of soricid populations to repeated fire and fuel reduction treatments in the southern Appalachian Mountains

Fuel hazards have increased in forests across the United States because of fire exclusion during the 20th century. Treatments used to reduce fuel buildup may affect wildlife, such as shrews, living on the forest floor, especially when treatments are applied repeatedly. From mid-May to mid-August 2006 and 2007, we used drift fences with pitfall traps to capture shrews in western North Carolina in 3 fuel reduction treatment areas [(1) twice-burned (2003 and 2006), (2) mechanical understory cut (2002), and (3) mechanical understory cut (2002) followed by 2 burns (2003 and 2006)] and a control. We captured 77% fewer southeastern shrews (Sorex longirostris) in mechanical + twice-burned treatment areas than in mechanical treatment areas in 2006, but southeastern shrew captures did not differ among treatment areas in 2007. Total shrew captures did not differ among treatment areas in either year. Decreases in leaf litter, duff depth, and canopy cover in mechanical + twice-burned treatment areas may have decreased ground-level moisture, thereby causing short-term declines in southeastern shrew captures. Prescribed fire or mechanical fuel reduction treatments in the southern Appalachian Mountains did not greatly affect shrew populations, though the combination of both treatments may negatively affect some shrew species, at least temporarily.     

Participation of phosphofructokinase,malate dehydrogenase and isocitrate dehydrogenase in capacitation and acrosome reaction of boar spermatozoa

The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well‐known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential–interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/10¹⁰ spermatozoa, the activity of NAD‐ and NADP‐dependent IDH was 0.111 ± 0.005 U/10¹⁰ and 2.22 ± 0.14 U/10¹⁰ spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/10¹⁰ spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.     

Protein Composition of Seminal Plasma in Fractionated Stallion Ejaculates

Seminal plasma (SP) contains several types of compounds derived from the epididymides and accessory glands. The aim of this study was to examine the protein composition of different ejaculate fractions. Trial I: fractionated ejaculates were collected from two normal and two subfertile stallions. Samples containing pre‐sperm fluid and the first sperm‐rich jets (HIGH‐1), the main sperm‐rich portion (HIGH‐2), the jets with low sperm concentrations (LOW), and a combined whole‐ejaculate (WE) sample was centrifuged, and the SP was filtered and frozen. A part of each SP sample was stored (5°C, 24 h) with spermatozoa from HIGH‐2 and skim milk extender. Sperm motility was evaluated after storage in extender mixed with the stallion’s own SP or SP from one of the other stallions (sperm from a normal stallion stored in SP from a subfertile stallion and vice versa). Protein composition was analysed using reverse‐phase liquid chromatography (RP‐HPLC), N‐terminal sequencing and mass spectrometry. The area‐under‐the‐curve (AUC) was used for quantitative comparison of proteins within fractions. Trial II: semen samples were collected from seven stallions. Fractions with the highest (HIGH) and lowest (LOW) sperm concentrations and WE samples were examined using SDS‐PAGE and densitometry. No significant differences emerged between fractions in the AUC‐values of the Horse Seminal Protein‐1 (HSP‐1) and HSP‐2 peaks, or the peak containing HSP‐3 and HSP‐4 (HSP‐3/4). Levels of HSP‐1, HSP‐2 and HSP‐3/4 were not significantly correlated with total sperm motility, progressive sperm motility or average path velocity after storage. Significant differences between ejaculate fractions in the amount of different protein groups present in SP were not found in Trial I; but in Trial II, the proteins in the 60–70 kDa range were more abundant in LOW than in HIGH and WE, indicating that this band contained proteins derived mainly from the seminal vesicles, which produce most of the SP in LOW.     

Short-term response of reptiles and amphibians to prescribed fire and mechanical fuel reduction in a southern Appalachian upland hardwood forest

We compared the effects of three fuel reduction techniques and a control on the relative abundance and richness of reptiles and amphibians using drift fence arrays with pitfall and funnel traps. Three replicate blocks were established at the Green River Game Land, Polk County, North Carolina. Each replicate block contained four experimental units that were each approximately 14 ha in size. Treatments were prescribed burn (B); mechanical understory reduction (M); mechanical + burn (MB); and controls (C). Mechanical treatments were conducted in winter 2001–2002, and prescribed burns in March 2003. Hot fires in MB killed about 25% of the trees, increasing canopy openness relative to controls. Leaf litter depth was reduced in B and MB after burning, but increased in M due to the addition of dead leaves during understory felling. The pre-treatment trapping period was short (15 August–10 October 2001) but established a baseline for post-treatment comparison. Post-treatment (2002–2004), traps were open nearly continuously May–September. We captured a total of 1308 species of 13 amphibians, and 335 reptiles of 13 species. The relative abundance of total salamanders, common salamander species, and total amphibians was not changed by the fuel reduction treatments. Total frogs and toads (anurans) and Bufo americanus were most abundant in B and MB; however, the proximity of breeding sites likely affected our results. Total reptile abundance and Sceloporus undulatus abundance were highest in MB after burning, but differed significantly only from B. Mean lizard abundance in MB was highest in 2004 and higher than in other treatments, but differences were not statistically significant. Our results indicate that a single application of the fuel reduction methods studied will not negatively affect amphibian or reptile abundance or diversity in southern Appalachian upland hardwood forest. Our study further suggests that high-intensity burning with heavy tree-kill, as in MB, can be used as a management tool to increase reptile abundance – particularly lizards – with no negative impact on amphibians, at least in the short-term.