Effectiveness of near-infrared spectroscopy as a non-invasive tool to discriminate spectral profiles of in vitro cultured oocytes from goats

Abstract Here, we aimed to discriminate between the spectral profiles of spent culture media after oocyte in vitro maturation (IVM) and culture (IVC) from goats of different ages subjected to repeated hormonal treatments. The profiles were discriminated using near infrared (NIR) spectroscopy combined with multivariate methods. A total of 19 goats (young = 10; old = 9) were subjected to serial hormonal stimulation (HS) with gonadotropins. Cumulus oophorus complexes (COCs) were collected using laparoscopic ovum pick-up (LOPU) and subjected to IVM and parthenogenetic activation. The initial embryos were subjected to IVC. Spent culture media were collected after oocyte IVM and on day 2 of IVC and analyzed using NIR spectroscopy. NIR spectral data were interpreted through chemometric methods, such as principle component analysis (PCA) and partial least square discriminant analysis (PLS-DA). The results of PCA analysis clearly showed a separation in the spectral profiles between the experimental groups (HS sessions; young and old animals) both after IVM and IVC. Overall, the main absorption bands were attributed to the C-H group second overtone, first overtone of O-H and N-H, and C-H combinations and may serve as molecular markers. On the other hand, the spectral data obtained using PLS-DA models provided a better classification of the groups. The results showed the possibility of discriminating young and old groups as well as the three HS sessions with high specificity, sensitivity, and accuracy using NIR spectra. Thus, the culture medium analysis using NIR spectroscopy combined with multivariate methods indicated the dissimilarities between the groups and provided an insight into the in vitro development of goat oocytes. This technique serves as an efficient, objective, rapid, and non-invasive method to discriminate spectral profiles.

Effectiveness of near-infrared spectroscopy as a non-invasive tool to discriminate spectral profiles of in vitro cultured oocytes from goats

Here, we aimed to discriminate between the spectral profiles of spent culture media after oocyte in vitro maturation (IVM) and culture (IVC) from goats of different ages subjected to repeated hormonal treatments. The profiles were discriminated using near infrared (NIR) spectroscopy combined with multivariate methods. A total of 19 goats (young = 10; old = 9) were subjected to serial hormonal stimulation (HS) with gonadotropins. Cumulus oophorus complexes (COCs) were collected using laparoscopic ovum pick-up (LOPU) and subjected to IVM and parthenogenetic activation. The initial embryos were subjected to IVC. Spent culture media were collected after oocyte IVM and on day 2 of IVC and analyzed using NIR spectroscopy. NIR spectral data were interpreted through chemometric methods, such as principle component analysis (PCA) and partial least square discriminant analysis (PLS-DA). The results of PCA analysis clearly showed a separation in the spectral profiles between the experimental groups (HS sessions; young and old animals) both after IVM and IVC. Overall, the main absorption bands were attributed to the C-H group second overtone, first overtone of O-H and N-H, and C-H combinations and may serve as molecular markers. On the other hand, the spectral data obtained using PLS-DA models provided a better classification of the groups. The results showed the possibility of discriminating young and old groups as well as the three HS sessions with high specificity, sensitivity, and accuracy using NIR spectra. Thus, the culture medium analysis using NIR spectroscopy combined with multivariate methods indicated the dissimilarities between the groups and provided an insight into the in vitro development of goat oocytes. This technique serves as an efficient, objective, rapid, and non-invasive method to discriminate spectral profiles.(AU)

Impact of hypotensive action of angiotensin converting enzyme inhibitor on ovarian-intraovarian blood flow and follicles development in goats hormonally stimulated with repeated FSH-ECG treatment

Background: Recent evidence shows that the renin-angiotensin system (RAS) participates in important reproductiveprocesses, such as steroidogenesis, folliculogenesis, oocyte maturation and ovulation. Several studies have proposed touse an angiotensin-converting enzyme (ACE) as a RAS modulator, aiming to improve reproductive efficiency, however,the presence of the main components of this system in reproductive tissues still need to be further investigated, since thephysiological functions seem to be species-specific. The aim of this study was to assess the impact of enalapril-maleate,an ACE inhibitor, during repeated gonadotropins treatment on ovarian blood flow and follicular development in goats.Materials, Methods & Results: Twenty Anglo-Nubian cross-bred goats were equally grouped according to parity (n= 10/group): nulliparous and multiparous parity. In each group, five animals were randomly selected to receive 0.4 mg.kg-1 ofenalapril-maleate during 11 days of estrus synchronization and gonadotropins treatments. The other animals received thesame volume of saline solution. Estrus synchronization of all goats was made by intramuscular ad-ministration of PGF2αanalog, followed 48 h later by intravaginal insertion of a controlled internal drug release device. Forty-eight h after devicewithdrawal, a single dose of 60 mg of FSH plus 300 UI of eCG was administered and repeated every 4 days to complete 3treatments. Transrectal ultrasonography was performed using pulsed and color Doppler to evaluate Doppler velocimetricsparameters of the ovarian artery and intraovarian blood flow, respec-tively, and B-mode real-time ultrasound scanner toevaluate the follicular development. In the females treated with enalapril-maleate was observed a significant reduction ofsystolic and diastolic peak, without difference according to parity. In addition, in the third session of hor-monal stimulation,only the groups (nulliparous and multiparous) not treated with...(AU)

Effect of supplementation of Aloe vera extracts in cold storage media and cryopreservation of domestic cat epididymal spermatozoa

This study evaluated the effect of the extract of Aloe vera at concentrations of 10% and 20% on the cryopreservation of sperm from the epididymis of domestic cats. Epididymal spermatozoa were recovered using the flotation technique and used in the treatments: control (TRIS-egg yolk at 20%), T10% (TRIS plus 10% of A. vera extract), and T20% (TRIS plus 20% of A. vera extract). The spermatozoa were subjected to 4ºC for 60 minutes, followed by 20 minutes in nitrogen vapors, and stored in a cryogenic cylinder. The samples were thawed at 37°C for 30 seconds. The sperm motility decreased (P0.05) during freezing; however, after thawing, it decreased (P0.05). The effect of the crude A. vera extract was not satisfactory on the cryopreservation of epididymal spermatozoa of domestic cats after thawing; although the motility of spermatozoa was similar to that found with the use of egg yolk, and it presented maintenance of the chromatin integrity. However, it is necessary to understand the action of the substances present in A. vera with the feline spermatozoa, well as the standardization and adjustment of physicochemical characteristics aiming at the future application of the vegetal extract.

Impact of hypotensive action of angiotensin converting enzyme inhibitor on ovarian-intraovarian blood flow and follicles development in goats hormonally stimulated with repeated FSH-ECG treatment

Background: Recent evidence shows that the renin-angiotensin system (RAS) participates in important reproductiveprocesses, such as steroidogenesis, folliculogenesis, oocyte maturation and ovulation. Several studies have proposed touse an angiotensin-converting enzyme (ACE) as a RAS modulator, aiming to improve reproductive efficiency, however,the presence of the main components of this system in reproductive tissues still need to be further investigated, since thephysiological functions seem to be species-specific. The aim of this study was to assess the impact of enalapril-maleate,an ACE inhibitor, during repeated gonadotropins treatment on ovarian blood flow and follicular development in goats.Materials, Methods & Results: Twenty Anglo-Nubian cross-bred goats were equally grouped according to parity (n= 10/group): nulliparous and multiparous parity. In each group, five animals were randomly selected to receive 0.4 mg.kg-1 ofenalapril-maleate during 11 days of estrus synchronization and gonadotropins treatments. The other animals received thesame volume of saline solution. Estrus synchronization of all goats was made by intramuscular ad-ministration of PGF2αanalog, followed 48 h later by intravaginal insertion of a controlled internal drug release device. Forty-eight h after devicewithdrawal, a single dose of 60 mg of FSH plus 300 UI of eCG was administered and repeated every 4 days to complete 3treatments. Transrectal ultrasonography was performed using pulsed and color Doppler to evaluate Doppler velocimetricsparameters of the ovarian artery and intraovarian blood flow, respec-tively, and B-mode real-time ultrasound scanner toevaluate the follicular development. In the females treated with enalapril-maleate was observed a significant reduction ofsystolic and diastolic peak, without difference according to parity. In addition, in the third session of hor-monal stimulation,only the groups (nulliparous and multiparous) not treated with...

Effect of supplementation of Aloe vera extracts in cold storage media and cryopreservation of domestic cat epididymal spermatozoa

This study evaluated the effect of the extract of Aloe vera at concentrations of 10% and 20% on the cryopreservation of sperm from the epididymis of domestic cats. Epididymal spermatozoa were recovered using the flotation technique and used in the treatments: control (TRIS-egg yolk at 20%), T10% (TRIS plus 10% of A. vera extract), and T20% (TRIS plus 20% of A. vera extract). The spermatozoa were subjected to 4ºC for 60 minutes, followed by 20 minutes in nitrogen vapors, and stored in a cryogenic cylinder. The samples were thawed at 37°C for 30 seconds. The sperm motility decreased (P<0.05) after thawing in the three treatments. Only the spermatozoa in the control treatment maintained post-thawing vigor. The viability of spermatozoa decreased in the treatments with A. vera (P<0.05). According to the hypoosmotic test, all treatments maintained the sperm membrane functionality (P>0.05) during freezing; however, after thawing, it decreased (P<0.05) in the T10% and T20% treatments. The morphology and chromatin condensation of spermatozoa did not differ, regardless of the treatments and time of evaluation (P>0.05). The effect of the crude A. vera extract was not satisfactory on the cryopreservation of epididymal spermatozoa of domestic cats after thawing; although the motility of spermatozoa was similar to that found with the use of egg yolk, and it presented maintenance of the chromatin integrity. However, it is necessary to understand the action of the substances present in A. vera with the feline spermatozoa, well as the standardization and adjustment of physicochemical characteristics aiming at the future application of the vegetal extract.(AU)

Espectroscopia NIR e análise multivariada aplicadas à identificação de grupos espectrais de células tronco derivadas do fluido amniótico de caprinos / NIR spectroscopy and multivariate analysis applied to the identification of spectrum groups of trunk cells derived from goats amniotic fluid

O objetivo deste trabalho foi desenvolver um modelo preditivo através de técnica multivariada para diferenciar meios de cultivo de células-tronco cultivadas in vitro e criopreservadas de acordo com os perfis de absorbância obtidas por NIR. Para tanto, foram coletados meios de cultivo de células-tronco oriundo do fluido amniótico de fetos caprinos, antes e após o processo de criopreservação por vitrificarão, e submetidos à análise pelo NIR. Foi possível estimar com alta acurácia o tratamento empregado nas amostras, gerando uma impressão digital dos meios de cultivo in vitro de células criopreservadas ou não.

The objective of this work was to develop a predictive model through a multivariate technique to differentiate culture media from stem cells cultured in vitro and cryopreserved according to the absorbance profiles obtained by NIR. For this purpose, culture media were collected from stem cells from the amniotic fluid of goat fetuses, before and after cryopreservation by vitrification, and submitted to NIR analysis. It was possible to estimate with high accuracy the treatment used in the samples, generating a fingerprint of in vitro culture media of cryopreserved cells or not.

Espectroscopia NIR e análise multivariada aplicadas à identificação de grupos espectrais de células tronco derivadas do fluido amniótico de caprinos / NIR spectroscopy and multivariate analysis applied to the identification of spectrum groups of trunk cells derived from goats amniotic fluid

O objetivo deste trabalho foi desenvolver um modelo preditivo através de técnica multivariada para diferenciar meios de cultivo de células-tronco cultivadas in vitro e criopreservadas de acordo com os perfis de absorbância obtidas por NIR. Para tanto, foram coletados meios de cultivo de células-tronco oriundo do fluido amniótico de fetos caprinos, antes e após o processo de criopreservação por vitrificarão, e submetidos à análise pelo NIR. Foi possível estimar com alta acurácia o tratamento empregado nas amostras, gerando uma impressão digital dos meios de cultivo in vitro de células criopreservadas ou não.(AU)

The objective of this work was to develop a predictive model through a multivariate technique to differentiate culture media from stem cells cultured in vitro and cryopreserved according to the absorbance profiles obtained by NIR. For this purpose, culture media were collected from stem cells from the amniotic fluid of goat fetuses, before and after cryopreservation by vitrification, and submitted to NIR analysis. It was possible to estimate with high accuracy the treatment used in the samples, generating a fingerprint of in vitro culture media of cryopreserved cells or not.(AU)

Valores hematológicos e bioquímicos de camundongos imunossuprimidos BALB/c NUDE E C57BL/6 SCID / Haematological and biochemical values of immunosuppressed BALB/c NUDE and C57BL/6 SCID mice

Background: The emergence of the NUDE and SCID immunosuppressed mice lineages generated knowledge on various mechanisms of lymphocyte maturation and human autoimmune diseases. Information on haematological and biochemical parameters of these lineages is still scarce, making it impossible to infer homeostasis by comparing data, or to detect genetic influences on the parameters for these species. Haematological and biochemical tests were carried out on Balb/c NUDE and C57BL/6 SCID mice of both sexes, aiming to analyse the presence of genetic influence on possible variations of such parameters and to verify reference values for both lineages.Materials, Methods & Results: One hundred and forty mice (Mus musculus) of the Balb/C NUDE and C57BL/6 SCID lineages were used in the present study. The animals were previously anesthetized, the blood collection procedure was performed by cardiac puncture and the samples were collected in the presence of heparin and intended for haematological and biochemical evaluation, under standardized conditions. The haematological evaluation consisted of red blood cell count, leukocyte counts, platelet counts, haematocrit, haemoglobin concentration, mean corpuscular volume (MCV), and mean corpuscular haemoglobin concentration (MCHC). The quantified biochemical parameters were: urea, creatinine, alanine aminotransaminase (ALT), aspartate aminotransaminase (AST) and alkaline phosphatase (ALP). While analysing the obtained data, it was possible to observe that only females presented divergences (P < 0.05) in the red blood cell series, in haemoglobin and in mean haemoglobin concentration (MCH). Regarding the analysis of the white blood cell series, females only presented differences (P < 0.05) in the leukocyte count. For males, there were variations (P < 0.05) in the counts of leukocytes, eosinophils, lymphocytes and neutrophils.[...]

Valores hematológicos e bioquímicos de camundongos imunossuprimidos BALB/c NUDE E C57BL/6 SCID / Haematological and biochemical values of immunosuppressed BALB/c NUDE and C57BL/6 SCID mice

Background: The emergence of the NUDE and SCID immunosuppressed mice lineages generated knowledge on various mechanisms of lymphocyte maturation and human autoimmune diseases. Information on haematological and biochemical parameters of these lineages is still scarce, making it impossible to infer homeostasis by comparing data, or to detect genetic influences on the parameters for these species. Haematological and biochemical tests were carried out on Balb/c NUDE and C57BL/6 SCID mice of both sexes, aiming to analyse the presence of genetic influence on possible variations of such parameters and to verify reference values for both lineages.Materials, Methods & Results: One hundred and forty mice (Mus musculus) of the Balb/C NUDE and C57BL/6 SCID lineages were used in the present study. The animals were previously anesthetized, the blood collection procedure was performed by cardiac puncture and the samples were collected in the presence of heparin and intended for haematological and biochemical evaluation, under standardized conditions. The haematological evaluation consisted of red blood cell count, leukocyte counts, platelet counts, haematocrit, haemoglobin concentration, mean corpuscular volume (MCV), and mean corpuscular haemoglobin concentration (MCHC). The quantified biochemical parameters were: urea, creatinine, alanine aminotransaminase (ALT), aspartate aminotransaminase (AST) and alkaline phosphatase (ALP). While analysing the obtained data, it was possible to observe that only females presented divergences (P < 0.05) in the red blood cell series, in haemoglobin and in mean haemoglobin concentration (MCH). Regarding the analysis of the white blood cell series, females only presented differences (P < 0.05) in the leukocyte count. For males, there were variations (P < 0.05) in the counts of leukocytes, eosinophils, lymphocytes and neutrophils.[...](AU)