Effect of γ-oryzanol on testicular degeneration induced by scrotal insulation in rams

Testicular degeneration is highly prevalent in ruminants reared in a tropical climate in Brazil. It is therefore necessary to assess semen quality periodically in this animals. The objective of this work was to define standards images (thermograms) by using infrared thermography (TIV), associated with computerized seminal evaluation (CASA) to predict testicular alterations in a non-invasive way. For this, 10 goat males were selected, and testis insulated to evaluate the seminal quality and testicular superficial temperature recorded in four different moments: before, during and early and later after insulation. Semen collections and rectal temperatures (TR) and superficial testis (TS) by TIV were assessed for 60 days after insulation. Data were expressed in media and standard errors and analysis of variation (ANOVA) with comparison between the moments using Tukey test at 5% of probability. Changes in seminal quality followed the same standard of changes in TS, with no influence of the latter one on rectal temperature. The mean testicular temperature increased more than 2 degrees above the normal mean testis temperature, with significant increasing (p <0.05) in the moment during in relation to the moments before and post insulation, and concomitantly reduction in seminal parameters and almost normal return to 60 days post insulation. These changes in testis surface temperatures associated with semen analysis allowed to get patterns images of testicular degeneration, permitting to predict by TIV testis alterations for later assess of seminal quality.

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265 TESTICULAR DEGENERATION AFFECTED PLASMA, ACROSOMAL AND MITOCHONDRIAL MEMBRANE INTEGRITY, AND DNA FRAGMENTATION IN RAM SPERMATOZOA

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab265 ◽

2015 ◽

Vol 27 (1) ◽

pp. 222

Author(s):

M. Bianchi Rodrigues Alves ◽

A. Furugen Cesar de Andrade ◽

R. Paes de Arruda ◽

L. Batissaco ◽

R. Lançoni ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Potential ◽

Dna Fragmentation ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Membrane Integrity ◽

Mitochondrial Potential ◽

Plasma Membrane Integrity ◽

Scrotal Insulation ◽

Testicular Degeneration

Testicular degeneration, an important cause of male infertility, adversely affects sperm motility and morphology. However, few studies describe effects on integrity of plasma and acrosomal membranes, mitochondrial membrane potential, and DNA fragmentation; therefore, they were evaluated in the present study. Testicular degeneration was induced in 17 White Dorper rams (scrotal insulation for 72 h). Semen was collected (artificial vagina) twice before insulation and twice thereafter (15-day intervals between post-insulation collections). Sperm motility and morphology were analysed by SCA software (Sperm Class Analyser®, MICROPTIC®, Barcelona, Spain) and differential interference contrast microscopy (DIC, model 80i, Nikon, Tokyo, Japan), respectively. Membrane integrity and potential were assessed with fluorescent probes: Hoescht 33342, propidium iodide, FITC-PSA, and JC-1 (Celeghini et al. 2010 Arq. Bras. Med. Vet. Zootec. 62, 536–543) and imaged with fluorescence microscopy (Nikon Model 80i, Nikon, Tokyo, Japan). Fragmentation of DNA was evaluated with a Halomax® kit (Halotech® DNA, Madrid, Spain). Data were analysed with Statview software (Stat View 1998, SAS Institute Inc., Cary, NC, USA). Data obtained from the periods (before × after insulation) were evaluated by analysis of variance (ANOVA) and means were compared using Tukey's test. Total motility (before: 87.53 ± 1.21%; after: 46.53 ± 4.46%) and progressive motility (before: 58.64 ± 2.00%; after: 31.33 ± 3.82%) were reduced (P < 0.01) by scrotal insulation, as were sperm major defects (before: 10.64 ± 1.65%; after: 54.30 ± 3.67%) and total defects (before: 20.50 ± 2.40%; after: 63.85 ± 3.41%; P < 0.0001). Sperm with intact plasma and acrosomal membranes and high mitochondrial potential (PIAIH) decreased (P < 0.0001) after insulation. In that regard, 53.19 ± 2.20 and 28.48 ± 3.48% of sperm were classified as PIAIH before v. after insulation, respectively. Furthermore, plasma membrane integrity, acrosome membrane integrity, and high mitochondrial potential were assessed independently. The quantity of plasma membrane integrity cells (before: 62.01 ± 2.07%; after: 33.92 ± 3.94%), acrosome membrane integrity cells (before: 57.17 ± 2.30%; after: 31.47 ± 3.77%), and high mitochondrial potential cells (before: 85.72 ± 1.42%; after: 57.28 ± 3.12%) were also reduced (P < 0.0001) after insulation. Likewise, DNA integrity decreased (P = 0.002) from 98.87 ± 0.26% before insulation to 91.88 ± 2.6% afterward. In conclusion, sperm plasma and acrosomal membrane integrity, mitochondrial membrane potential, and DNA fragmentation were adversely affected by testicular degeneration in rams induced by scrotal insulation.Research was supported by FAPESP process 2012/00040-0 and 2011/16744-3.

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17 TESTICULAR HISTOPATHOLOGICAL CHARACTERISTICS OF RAMS TREATED WITH LOW-LEVEL LASER THERAPY: PRELIMINARY RESULTS

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab17 ◽

2014 ◽

Vol 26 (1) ◽

pp. 122

Author(s):

M. R. Bianchi-Alves ◽

E. C. C. Celeghini ◽

R. P. de Arruda ◽

A. F. C. De Andrade ◽

L. Batissaco ◽

...

Keyword(s):

Cumulative Dose ◽

Treatment Effect ◽

Seminiferous Tubule ◽

Low Level Laser Therapy ◽

Seminiferous Tubules ◽

Level Laser ◽

Group 2 ◽

Scrotal Insulation ◽

Testicular Degeneration ◽

Group 1

Testicular degeneration is caused by the loss of testicular thermoregulation induced by high environmental temperature, reproductive disruptions, fever, and others processes. Low-level laser therapy (LLLT) is an important tool to induce cellular proliferation and to stimulate mitosis. This biostimulatory effect was observed in the seminiferous epithelium of rats treated with LLLT, obtaining better results using a cumulative dose of 28.05 J cm–2 rather than 46.80 J cm–2 (Taha and Valojerdi 2004 Lasers Surg. Med. 34, 352–359). Thus, the aim of this study was to evaluate the effect of different LLLT protocols on testes histopathological characteristics of rams submitted to scrotal insulation for the induction of the testicular degeneration. For this, 6 rams were divided in 3 groups: (1) control, without LLLT treatment (n = 2); (2) LLLT treatment with a cumulative dose of 28 J cm–2 (n = 2); and (3) LLLT treatment with a cumulative dose of 56 J cm–2 (n = 2). Treatment was performed once a day and repeated every 48 h during a 15-day interval in the treated groups (2 and 3). The output power used was the same for the treated groups (30 mW). Scrotal insulation was done in all rams at 72 h before the beginning of the treatment period. The rams were castrated after 35 days of scrotal insulation. Both testes were divided in 3 parts: ventral, medium, and dorsal. The testes segments were fixed using Bouin fluid and stained using hematoxylin-eosin. Analyses were performed by light microscopy (model 80i; Nikon, Tokyo, Japan) using the NIS-Elements AR® to measure the lumen of the seminiferous tubules and the seminiferous tubules total area. Ten different areas of each testicular segment were analysed and, in each area, 2 seminiferous tubules were chosen randomly. For this analysis, the percentage of the lumen area of the seminiferous tubule in relation to the total area of the seminiferous tubule (lumen/lumen + tubule) was determined. In parallel, degree of degeneration was classified by blind analysis, as light, moderate, or severe, in each testicular segment. The data was analysed employing SAS (SAS Institute Inc., Cary, NC, USA). The treatment effect was tested using PROC GLM (ANOVA). Tukey's test was used to compare the averages. A treatment effect (P < 0.001) was observed. Group 2 (14.55 ± 2.08%) presented a lower percentage of lumen than did group 1 (36.78 ± 3%) and 3 (30.98 ± 2.24%). The degeneration degree of ram testes of group 1 was classified as moderate for 1 ram and severe for the other, whereas, in group 2, it was classified as light for both rams. For group 3, it was classified as moderate for both rams. Thus, it is possible to conclude that LLLT treatment is efficient, and that the cumulative dose of 28 J cm–2 is more efficient than the cumulative dose of 56 Jcm–2, a corroboration of previous results in rats (Ibid.). However, the results are preliminary and more studies are being done by our group. The authors acknowledge FAPESP processes 2012/00040-0 and 2011/16744-3.

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