Abstract The aim of the present study was to evaluate semen cryopreservation with ACP-Lact® diluent, which consists of coconut water powder (ACP) added to goat milk powder. After thawing, the samples were evaluated for sperm kinetics, membrane evaluation and in vivo insemination. For cryopreservation, a pool was made with the ejaculate of six goats, diluted in four equal aliquots for the respective treatments: T1 (ACP-Lact®); T2 (ACP-Lact® 50%); T3 (ACP + 2.5% egg yolk) and T4 (Tris + 2.5% egg yolk). After dilution of the treatments, the samples were placed in 0.5 ml straws and chilled at a rate of -1.07°C/min. After reaching 4°C and stabilizing for one hour, the straws were placed in nitrogen vapour at -60°C for 15 minutes and then immersed in liquid nitrogen (-196ºC). The straws were thawed in a 37°C water bath and kinetic assessments were performed immediately using a computerized semen analysis program (CSA), viability (EN), membrane functionality (HOST), mitochondrial activity (DAB) and DNA integrity assessment of spermatozoa. For the in vivo experiment, ten goats were inseminated, divided into two groups of five goats each, G1 inseminated with ACP-Lact® and G2 with ACP, by fixed-time artificial insemination (FTAI). Regarding the kinetic parameters, the ACP-Lact® treatment showed higher progressive motility (PM) and sperm velocity than the other treatments (36.77%). In the VSL parameter the ACP-Lact diluent was superior to ACP and Tris. In viability the treatment with ACP-Lact® was superior to the treatment with Tris, 95% and 83% respectively. In FTAI two goats were born out of the 5 goats inseminated with ACP-Lact®. It was concluded that the use of ACP-Lact® for cryopreservation of caprine semen is efficient in maintaining seminal parameters during thawing in vitro and in vivo and proved to be a good alternative extender for the caprine species. ACPLact® ACPLact Lact® Lact kinetics T (ACP-Lact®) (ACP-Lact 50% 50 50%) 25 2 2.5 yolk Tris yolk. . 05 0 0. 1.07°C/min. 107Cmin Cmin 1 07 C min -1.07°C/min 4C 4 hour 60°C 60C 60 196ºC. 196ºC ºC (-196ºC) 37C 37 CSA, CSA , (CSA) EN, EN (EN) HOST, HOST (HOST) DAB (DAB spermatozoa experiment each G fixedtime fixed time FTAI. (FTAI) PM (PM 36.77%. 3677 36.77% 36 77 (36.77%) 95 83 respectively ACPLact®. Lact®. species 2. 1.07°C/min 6 (-196ºC 3 (CSA (EN (HOST (FTAI 367 36.77 7 (36.77% 9 8 36.7 (36.77 36. (36.7 (36. (36 (3 (

Abstract To assist the reproductive management of tambaqui (Colossoma macropomum) males in laboratory and commercial fish farming, a linear regression model was obtained from concentration curves using the spectrophotometric method. Twenty-two tambaqui males with an average age of three years old were selected and divided into two groups containing 11 animals each. Both groups alternately received a single dose of carp pituitary extract (CPE; 2.0 mg/kg body weight, intracoelomic). Sperm was collected 14 h after hormonal treatment and diluted (1:4000; sperm:formaldehyde saline). The concentration was estimated by counting spermatozoa in a Neubauer chamber and by using a spectrophotometer (λ=540 nm). Individual sperm concentration ranged from 11.40 to 71.13 × 109 sperm/mL. The degree of transmittance ranged from 62.1% to 95.0%. There was a significant correlation (r2 = 0.966; p < 0.0001) between sperm concentration analyzed in a Neubauer chamber and transmittance at 540 nm. Analysis by spectrophotometry and the prediction provided by the equation Y=100.293 - 0.509X proved to be an efficient and fast method for estimating sperm concentration in tambaqui and can be used in routine procedures in artificial fish reproduction laboratories. Colossoma macropomum farming Twentytwo Twenty 1 each CPE (CPE 20 2 0 2. mgkg mg kg weight intracoelomic. intracoelomic . intracoelomic) 14000 4000 (1:4000 spermformaldehyde formaldehyde saline. saline saline) λ=540 λ540 λ (λ=54 nm nm) 1140 40 11.4 7113 71 13 71.1 10 spermmL mL sperm/mL 621 62 62.1 950 95 95.0% r2 r (r 0.966 0966 966 0.0001 00001 0001 54 Y100293 Y 100 293 Y=100.29 0509X X 509X laboratories 1400 400 (1:400 λ=54 λ54 (λ=5 114 4 11. 711 7 71. 6 62. 9 95.0 0.96 096 96 0.000 0000 000 5 Y10029 29 Y=100.2 140 (1:40 λ=5 λ5 (λ= 95. 0.9 09 0.00 00 Y1002 Y=100. (1:4 λ= (λ 0. 0.0 Y100 Y=100 (1: Y10 Y=10 (1 Y1 Y=1 ( Y=

Resumo Visando auxiliar o manejo reprodutivo de machos de tambaqui (Colossoma macropomum) em piscicultura de laboratório e comercial, obteve-se um modelo de regressão linear a partir de curvas de concentração por método espectrofotométrico. Foram selecionados 22 machos de tambaqui com idade média de três anos. Eles foram divididos em dois grupos contendo 11 animais cada. Ambos os grupos receberam alternadamente uma única dose de extrato de hipófise de carpa (EHC; 2,0 mg/kg de peso corporal, intracelomático). O esperma foi coletado 14 horas após o tratamento hormonal e diluído (1:4000; esperma: solução salina formaldeído). A concentração foi estimada por contagem de espermatozoides em câmara de Neubauer e por espectrofotômetro (λ=540 nm). A concentração espermática individual variou de 11,40 a 71,13 × 109 espermatozoides/mL. O grau de transmitância variou de 62,1 a 95,0%. Houve correlação significativa (r2 = 0,966; p < 0,0001) entre a concentração espermática analisada em câmara de Neubauer e a transmitância em 540 nm. A análise por espectrofotometria e a predição pela equação Y=100,293 - 0,509X mostrou ser um método eficiente e rápido para estimar a concentração espermática de tambaqui, podendo ser utilizado em procedimentos de rotina em laboratórios de reprodução artificial de peixes. Colossoma macropomum comercial obtevese obteve se espectrofotométrico 2 anos 1 cada EHC (EHC 20 0 2, mgkg mg kg corporal intracelomático. intracelomático . intracelomático) 14000 4000 (1:4000 formaldeído. formaldeído formaldeído) λ=540 λ540 λ (λ=54 nm nm) 1140 40 11,4 7113 71 13 71,1 10 espermatozoidesmL mL espermatozoides/mL 621 62 62, 950 95 95,0% r2 r (r 0,966 0966 966 0,0001 00001 0001 54 Y100293 Y 100 293 Y=100,29 0509X X 509X peixes 1400 400 (1:400 λ=54 λ54 (λ=5 114 4 11, 711 7 71, 6 9 95,0 0,96 096 96 0,000 0000 000 5 Y10029 29 Y=100,2 140 (1:40 λ=5 λ5 (λ= 95, 0,9 09 0,00 00 Y1002 Y=100, (1:4 λ= (λ 0, 0,0 Y100 Y=100 (1: Y10 Y=10 (1 Y1 Y=1 ( Y=

Abstract Effects were assessed of the dilutants TRIS and ACP - 101c® with the addition of different guinea fowl (Numida meleagris) egg yolk concentrations. Fifteen ejaculates were collected from five goats of the Anglo Nubian breed. The ejaculates were pooled and then divided into 12 groups, two control groups (GC1 TRIS, with 2.5% Gallus gallus domesticus hen egg yolk GOGD), (GC2 Control Group ACP - 101c®, with the addition of 2.5% Gallus gallus domesticus hen egg yolk GOGD) and ten experimental groups (EG), containing TRIS and ACP added with different concentrations of egg yolk from guinea hen (Numida meleagris) (TRIS 2,5% GONM; TRIS 5% GONM; TRIS 10% GONM; TRIS 15% GONM; TRIS 20% GONM; ACP® 2,5% GONM; ACP® 5% GONM; ACP® 10% GONM; ACP® 15% GONM; ACP® 20% GONM). Then cryopreservation was carried out and the samples stored in liquid nitrogen (-196 °C). After seven days, the samples were thawed and assessed for spermatic kinetics, immunofluorescence and sperm morphology. Analysis of GOMN by the CASA system showed that the various parameters were similar to those of GOGD (P>0.05). The membrane integrity, mitochondrial potential and the acrosome were not influenced by the treatment (P>0.05) nor by the dilutant used for cryopreservation (P>0.05). The spermatic morphology was also preserved by the different GOGD and GONM concentrations in the ACP® and TRIS dilutants, with no statistically significant differences (P<0.05). It was concluded that Numida meleagris egg yolk, as external membrane cryoproctant added to the dilutants ACP-101c® and TRIS, improved goat semen quality.

Abstract The objective of this study was to test the efficiency of powdered coconut water (ACP-406®) base-medium without or with the addition of supplements on in vitro culture of isolated goat secondary follicles. Follicles were cultured for 18 days in α-MEM or in ACP-406®, both without supplements (referred to as α-MEM and ACP, respectively), or both supplemented with BSA, insulin, transferrin, selenium, glutamine, hypoxanthine, and ascorbic acid (referred to as α-MEM+ and ACP+). Follicular morphology, antrum formation, follicular and oocyte diameter, levels of glutathione (GSH), and chromatin configuration after in vitro maturation were evaluated. At the end of culture, ACP-406® base-medium (without or with supplements) showed a higher (P < 0.05) percentage of normal follicles than α-MEM (without or with supplements). Antrum formation was similar among α-MEM+, ACP and ACP+, and significantly higher than α-MEM without supplements. The follicular diameter was greater in ACP+ than α-MEM, and similar to other treatments. Moreover, fully and daily grown rates were higher (P < 0.05) in ACP-406® base-medium (without or with supplements) than α-MEM (without or with supplements). Levels of GSH were similar between ACP+ and α-MEM+ treatments. Both ACP+ and α-MEM+ allowed meiotic resumption without a significant difference between the two groups. In conclusion, supplemented ACP-406® base-medium maintained follicular survival and promoted the development as well as meiotic resumption of isolated goat secondary follicles cultured in vitro for 18 days.

O objetivo deste trabalho foi avaliar o diluente ACP-102c na criopreservação do sêmen ovino em comparação com o diluidor tris-glicose-gema (TRIS) e o sêmen fresco. Foram coletados 48 ejaculados de quatro ovinos, sendo tomadas duas alíquotas por ejaculado para diluição e criopreservação em ACP-102c ou TRIS e uma terceira alíquota utilizada para análise do sêmen fresco. O sêmen fresco e o criopreservado em ambos os diluidores foram avaliados para viabilidade, integridade de membrana plasmática e acrossomal, teste hiposmótico, fragmentação do DNA e de motilidade espermática. Após descongelamento, ambos os diluidores não diferiram para viabilidade espermática, integridade de membrana plasmática e acrossomal, fragmentação de DNA e nas variáveis quantitativas e qualitativas de velocidade espermática, mas diferiram no teste hiposmótico, motilidade total e progressiva e amplitude lateral da cabeça, bem como em todas as variáveis de motilidade avaliadas, exceto linearidade e progressividade, após duas horas de incubação à 37 ºC. Houve variabilidade entre reprodutores na motilidade total e progressiva do sêmen criopreservado em ACP-102c após descongelamento. O diluidor ACP-102c conferiu menor proteção aos espermatozoides ovinos contra danos do congelamento quando comparado ao TRIS, mas o aprimoramento de sua formulação e protocolos mais adequados de congelação poderão torná-lo uma alternativa na congelação do sêmen ovino.

The aim of this study was to evaluate the ACP-102c extender in the cryopreservation of ram semen compared to tris-glucose-egg yolk (TRIS) extender and fresh semen. Forty-eight ejaculates were collected from four rams and two aliquots per ejaculate were taken for dilution and cryopreservation in ACP-102c or TRIS and a third aliquot used for the fresh semen analysis. Either the fresh semen and cryopreserved in both extenders were evaluated for viability, integrity of plasma and acrosomal membrane, hypoosmotic swelling test, DNA fragmentation and sperm motility. The extenders did not differ for sperm viability, acrosome and plasma membrane integrity, DNA fragmentation and quantitative and qualitative parameters of sperm velocity after thawing, but differed in hypoosmotic swelling test, total and progressive motility and lateral extent of the head as well as in all motility parameters evaluated (except linearity and straightness) after two hours of incubation at 37 ºC. There was variability among rams in total and progressive motility of semen cryopreserved in ACP-102c after thawing. The ACP-102c extender showed less protection in the cryopreservation of ram sperm when compared to TRIS, but the improvement in its formulation and freezing protocols may make it an alternative to freezing ram semen.