Assisted reproduction using frozen-thawed semen has practical advantages, although cryopreservation is detrimental to sperm fertility in most mammals. We examined the influence of cryopreservation and reactive oxygen species (ROS) on ram sperm DNA stability (using SCSA), lipid peroxidation (LPO), chlortetracycline fluorescence (CTC) patterns, motility and viability. In Experiment 1, DNA integrity, LPO, CTC, motility and viability tests were performed on fresh and cryopreserved sperm after 0, 6, and 24 hr in synthetic oviductal fluid (SOF). In Experiment 2, fresh sperm were incubated in serum-free SOF (SOF-S; 1, 4, and 24 hr) with 0, 50, 150, or 300 microM H2O2 then assayed. Cryopreservation increased the percentage of sperm with a high DNA fragmentation index (%DFI), decreased the percentages of motile and viable sperm at thawing (0 hr), but did not affect LPO. H2O2 (150 or 300 microM) increased %DFI after 24 hr. LPO or sperm viability were not affected by H2O2, although most motility parameters decreased. H2O2 decreased the percentage of chlortetracycline pattern F sperm at 4 hr and increased the percentage of acrosome-reacted sperm (pattern AR) after 1 hr. Pooled data of Experiment 2 showed LPO was positively correlated with SCSA (r = 0.29 to r = 0.59; P < 0.05 to P < 0.01), while most motility parameters and the percentage of viable sperm were negatively correlated with LPO (r = -0.30 to r = -0.38; P < 0.05 to P < 0.01). LPO was positively correlated with the percentage of pattern AR sperm (r = 0.33; P < 0.01). Cryopreservation and H2O2 promote DNA instability in ram sperm, though motility is a more sensitive indicator of oxidative stress than the other parameters investigated.

ABSTRACT:We hypothesized that cryopreservation and incubation in conditions that mimic the female genital tract following insemination increases the susceptibility of ram sperm DNA to denaturation. Ram sperm samples (n ϭ 12) underwent the sperm chromatin structure assay (SCSA) and semen quality tests, including motility parameters, viability, and chlortetracycline fluorescence (CTC) patterns. We also assessed correlations between SCSA variables and semen quality parameters. Analyses were performed for both fresh and cryopreserved samples at 0, 3, and 20 hours of incubation in synthetic oviductal fluid (SOF; 39ЊC, 5% CO 2 ). The SCSA variables, mean alpha t (X␣ t ) and standard deviation of alpha t (SD␣ t ), were higher because of cryopreservation (P Ͻ .05, P Ͻ .001, respectively) after 20 hours in SOF. For both fresh and frozen spermatozoa, SCSA values (X␣ t , SD␣ t , and the percentage of cells outside the main population of ␣ t [%COMP␣ t ]) increased during incubation in SOF. Motility was negatively correlated with both SD␣ t and %COM-P␣ t , ranging from Ϫ0.39 (P Ͻ .01) to Ϫ0.59 (P Ͻ .001) for both fresh and cryopreserved semen; viability also was negatively correlated with X␣ t , SD␣ t , or %COMP␣ t (Ϫ0.36; P Ͻ .05, Ϫ.40 and -.46; P Ͻ .01, respectively) in fresh semen. The %COMP␣ t was positively correlated to the percentage of CTC pattern AR (P Ͻ .001) and negatively correlated to the percentages of patterns F and B (Ϫ0.33 to Ϫ0.60, P Ͻ .05 to P Ͻ .001). Variation among ejaculates within ram was observed (P Ͻ .01). Cryopreservation clearly facilitates DNA damage in physiological conditions. The low to moderate correlations between SCSA variables and classical semen quality parameters indicate that the SCSA provides additional information to standard tests for evaluating ram sperm quality.

The effect of dietary orange peel (OPE) and tomato pomace extract (TPE) supplementations on growth performance, plasma biochemicals, carcass characteristics and antioxidant status of growing male rabbits were investigated. A total of 96 rabbits (5 weeks old) were distributed into four groups. The first group received untreated pelleted diet (control). The second group was fed a diet containing ascorbic acid (AA; 1.0 g/kg diet), while the third and fourth groups consumed diets supplemented with 200 gm of OPE or (TPE, respectively. Our results indicated that OPE and TPE contained 59, 14.03 mg ascorbic acid/100 g DM, respectively. Growth performance, except feed conversion ratio, and carcass weight were improved by dietary supplementations. Dietary supplementations decreased kidneys, abdominal, back fats and ether extract of meat. Plasma protein and globulin levels were high in rabbits fed AA and TPE-supplemented diets. Low plasma total cholesterol and LDL-cholesterol concentrations were observed in rabbits fed the supplemented diets. Plasma AA was increased in rabbits fed AA and OPE-supplemented diets. Rabbits fed OPE and TPE-supplemented diets had great SOD activity. The best economic efficiency was recorded by rabbits fed the supplemented diets. Dietary supplementations of OPE and TPE could effectively improve growth performance, antioxidative status, modulate AA level in plasma and meat and lower plasma total cholesterol and LDL.

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