WO2006130884A2 - Medium for improving spermatozoa's vitality of domestic mammalian males and man - Google Patents
Medium for improving spermatozoa's vitality of domestic mammalian males and man Download PDFInfo
- Publication number
- WO2006130884A2 WO2006130884A2 PCT/YU2006/000010 YU2006000010W WO2006130884A2 WO 2006130884 A2 WO2006130884 A2 WO 2006130884A2 YU 2006000010 W YU2006000010 W YU 2006000010W WO 2006130884 A2 WO2006130884 A2 WO 2006130884A2
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- WO
- WIPO (PCT)
- Prior art keywords
- medium
- sperm
- spermatozoa
- humans
- viability
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
Definitions
- the sperm In order to accomplish its physiological function - fertilization, the sperm is usually characterized by: active progressive motility, power of reproduction and its normal morphology, i.e., absence of chromosomal aberrations in the spermatozoal nuclei. Additionally, they are also characterized by dual cell metabolism - aerobic and anaerobic, which is highly important for the role performed by the sex cells. Otherwise, the spermatozoa are highly specialized cells, carriers of the genetic material to the future generations.
- the male sperm - spermatozoa most commonly plays a major role.
- the former is obvious in the insemination technology, however also in natural mating of the animals, i.e. humans (coitus).
- the most common reason is poor motility of the spermatozoa, i.e., significantly reduced time of their survival in in vitro settings, and their pertinent penetration power.
- sex cells express in such sperm the so-called "ant-like" movements, particularly in male animals (and humans) affected by urogenital infections. Due to such problems, cryo-technological processing procedure of the semen is highly unsuccessful, being also unreliable upon natural mating (coitus). It becomes particularly obvious upon control of its quality (motility and viability) subsequent to the cryo-technological procedure or upon the control of the native, that is, frozen sperm of the domestic mammals and humans.
- buffer composition e.g. soluble salts of alkaline metals (Na/K), that is, salts of earth-alkaline metals (Mg/Ca), tris, egg-yolk buffered with sodium citrate, boiled milk, etc.
- Na/K alkaline metals
- Mg/Ca earth-alkaline metals
- tris egg-yolk buffered with sodium citrate, boiled milk, etc.
- the invention i.e., medium
- the invention is mixed with the sample of the native sperm of the domestic male animal, i.e., human sperm and/or with the same sperm samples of the given males that are previously frozen. Semen doses are thawed at 35-36°C for 20 seconds.
- hexose sugars particularly glucose, mannose, galactose and fructose among all hexoses.
- pentoses hexose and arabinose are preferential.
- Monosaccharides may be classified into: heptose, hexose, pentose, tetrose, triose, and glucose. The components were in the mix ratios: a) 44:36:20; b) 45:30:25; c) 33.33:33.33:33,333.
- the most preferential medium solution is prepared in distilled water, while solution pH ranges between 6.75 and 7.75, which gradually inclines from acidity to alkalinity.
- the composition of the medium is the following:
- pH calibration is performed with pH-meter using standard buffers (Merck- Germany) with their pH values ranging between 4.00 (acidity) 7.00 and (alkaline) 10.00. Adjustment of pH value is performed on "Iskra", type M. A. 5705 apparatus, Slovenia. Medium pH value is adjusted with 10% phosphoric acid and 1/M potassium hydroxide, depending on whether acid and/or base environment was desired. The medium is tested on the domestic mammal and human semen in laboratory conditions, primarily using sperm of bulls affected with urogenital infections, i.e. from healthy heads (model/method - experimental).
- composition ratio of components (medium) used in each experiment was a:b:c both in experimental conditions and in practice, being: KH 2 PO 4 H 12 +Na 2 HPO 4 x 12H 2 O + O 6 x H 2 O, with pH being 6.75, i.e., 7.10.
- the experiment was performed in the laboratory using special light counter-phase microscope specially adjusted to these needs.
- Male animal sperm was applied drop-wise onto the slide glass to be subsequently mixed with the above- mentioned medium.
- the mixed sperm and a drop of the medium was covered with cover glass in order to enable monitoring of favorable/unfavorable effects (influence) of the medium on the spermatozoa under the microscope, i.e., their motility and viability.
- the results of the tests are tabularly presented within the Description section.
- the above-mentioned studies are carried out on the sperm samples obtained from both infected and healthy males.
- Composition of the medium was the following: KH 2 PO 4 +Na 2 HPO 4 x 12H 2 O + CgH 12 O 6 x H 2 O in a:b:c ratio.
- Medium composition was the following: KH 2 PO 4 +Na 2 HPO 4 x 12H 2 O x C 6 Hi 2 O 6 x H 2 O.
- the medium (pH 6.75) was applied intracervically simultaneously or before insemination of the cows. Naturally, the cows were in estrus at the time. The insemination was performed john artis, and thus bull semen dose used for insemination was mixed with the medium in the cervical canal, i.e., with estral mucus of the cows.
- Composition of the medium was the following: KH 2 PO 4 +Na 2 HPO 4 x 12H 2 O + C 6 H 12 O 6 x H 2 O in a:b:c ratio
- the medium (pH 7.10) was also studied on the sperm of the breeding bulls using both native and frozen sperm, as well as the influence of the medium on the gravidity (conception) success rate (%) achieved in the inseminated cows.
- the medium exerts its actions on the spermatozoa:
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- General Health & Medical Sciences (AREA)
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Abstract
The medium contains three components a, b and c. Upon addition to the native or frozen animal or human sperm it has highly favorable effects on the spermatozoa, i.e., their viability, survival period, progressive movement and their inherent penetration capacity, that is, on the sperm metabolism. The former is particularly true for poorly mobile sperm of the domestic animals and humans, and thus it influences their progressive motility, that is, their fertilization capacity. In other words, better fertilization rate of the inseminated animals and/or fertility of childless couples, i.e., conception by insemination or via the natural route (coitus). Namely, the medium may be used in two ways: a) in the course of insemination before inoculation of the sperm dose and/or immediately before natural mating/coitus; b) with previous treatment of the nature sperm in 1 : 1 ratio with the proposed medium, and such diluted animal or human sperm is subsequently subjected to further technological processing, deep freezing or it is used diluted in the native condition.
Description
MEDIUM FORIMPROVING SPERMATOZOA'S VITALITY OF DOMESTIC
MAMMALIAN MALES AND MAN
FILED OF INVENTION: REPRODUCTION (FIELD) - FERTILITY
Sexual reproduction is the basic characteristic of the multicellular living world. It cannot be attained without sperm (sex cells) of mails. In order to accomplish its physiological function - fertilization, the sperm is usually characterized by: active progressive motility, power of reproduction and its normal morphology, i.e., absence of chromosomal aberrations in the spermatozoal nuclei. Additionally, they are also characterized by dual cell metabolism - aerobic and anaerobic, which is highly important for the role performed by the sex cells. Otherwise, the spermatozoa are highly specialized cells, carriers of the genetic material to the future generations.
TECHNICAL ISSUE
The problem of spermatology (reproduction)
Within a range of mutually interwoven factors leading to disturbance of the reproduction in the domestic mammals and humans, the male sperm - spermatozoa most commonly plays a major role. The former is obvious in the insemination technology, however also in natural mating of the animals, i.e. humans (coitus). The most common reason is poor motility of the spermatozoa, i.e., significantly reduced time of their survival in in vitro settings, and their pertinent penetration power. Additionally, sex cells express in such sperm the so-called "ant-like" movements, particularly in male animals (and humans) affected by urogenital infections. Due to such problems, cryo-technological processing procedure of the semen is highly unsuccessful, being also unreliable upon natural mating (coitus). It becomes particularly obvious upon control of its quality (motility and viability) subsequent to the cryo-technological procedure or upon the control of the native, that is, frozen sperm of the domestic mammals and humans.
BACKGROUND OF INVENTION IN SPERMATOLOG
In order to resolve such situation in the spermatology (reproduction) at least to the certain extent, or in another words, in order to preserve viability and progressive motility of the spermatozoa, i.e., their power of reproduction, numerous diluents and/ or extenders of the native sperm of the domestic animals and humans were applied. With different variation of buffer composition, (e.g. soluble salts of alkaline metals (Na/K), that is, salts of earth-alkaline metals (Mg/Ca), tris, egg-yolk buffered with sodium citrate, boiled milk, etc.) according to formulations developed by numerous authors reported in professional-medical literature. Spermatozoal viability and motility of the domestic mammals and humans are basic characteristic of their reproductive power, particularly after their freezing. Numerous authors, both in the field of animal and human spermatology, have studied possibilities of preservation of viability and motility of the male sex cells for the prolonged period of time, as well as diluents and/or implementers of domestic animal and human sperm, which also prolong viability, i.e., motility of the sex cells in in vitro conditions, for the purpose of achieving of the best possible fertility in useful domestic mammals, i.e., in infertile human marriages. With this respect, Polge (1949), Rowson & Polge (1950) are the most outstanding authors in the field of the animal spermatology, as well as Sherman 1964, Bush (1991) and Norman-Johnson (1971) in human spermatology and numerous other researchers in the field of reproductions.
DETAILED DESCRIPTION OF INVENTION
The invention (i.e., medium) is mixed with the sample of the native sperm of the domestic male animal, i.e., human sperm and/or with the same sperm samples of the given males that are previously frozen. Semen doses are thawed at 35-36°C for 20 seconds. The medium is composed of three components: a) primary Na phosphate; b) secondary K phosphate and c) also one of monosaccharides (CH2O) x (triose, tetrose, hexose, pentose, hexose) depending on x value (x = 3,4,5,6,7). The most suitable are hexose sugars, particularly glucose, mannose, galactose and fructose among all hexoses. As for pentoses, hexose and arabinose are preferential. Monosaccharides may be classified into: heptose, hexose, pentose, tetrose, triose, and glucose.
The components were in the mix ratios: a) 44:36:20; b) 45:30:25; c) 33.33:33.33:33,333. The most preferential medium solution is prepared in distilled water, while solution pH ranges between 6.75 and 7.75, which gradually inclines from acidity to alkalinity. These medium components are completely compatible (tolerant) with the cells in the organism, particularly with the male sex cells which are produced in the testicles of male animals and/or humans (tubuli contorti s. seminiferis) via the process of spermatogenesis. The composition of the medium is the following:
KH2PCM-Na2HPO4 x 12H2O/ + one (CH2O)x (C6H12O6XH2O), which are previously dissolved in 100 cc. M/ 15 buffered distilled water as previously stated.
Medium pH adjustment
pH calibration is performed with pH-meter using standard buffers (Merck- Germany) with their pH values ranging between 4.00 (acidity) 7.00 and (alkaline) 10.00. Adjustment of pH value is performed on "Iskra", type M. A. 5705 apparatus, Slovenia. Medium pH value is adjusted with 10% phosphoric acid and 1/M potassium hydroxide, depending on whether acid and/or base environment was desired. The medium is tested on the domestic mammal and human semen in laboratory conditions, primarily using sperm of bulls affected with urogenital infections, i.e. from healthy heads (model/method - experimental). Composition, ratio of components (medium) used in each experiment was a:b:c both in experimental conditions and in practice, being: KH2PO4H12 +Na2HPO4 x 12H2O + O6 x H2O, with pH being 6.75, i.e., 7.10.
The experiment was performed in the laboratory using special light counter-phase microscope specially adjusted to these needs. The experiment is carried out in the ambient of isothermy (T=36 - 38.5°C) which imitated body temperature, i.e., natural environment of male domestic animal and human sperm. Male animal sperm was applied drop-wise onto the slide glass to be subsequently mixed with the above- mentioned medium. The mixed sperm and a drop of the medium was covered with cover glass in order to enable monitoring of favorable/unfavorable effects (influence) of the medium on the spermatozoa under the microscope, i.e., their motility and viability. The results of the tests are tabularly presented within the Description
section. The above-mentioned studies are carried out on the sperm samples obtained from both infected and healthy males.
The experiments were performed in vitro in order to evidence advantages of the favorable effects of the medium on the viability and survival of the spermatozoa, i.e., their power of reproduction.
A) LABORATORY ANALYSES OF THE MEDIUM (pH 6.75) IN BULL SPERM AFFECTED BY URINARY INFECTION
Composition of the medium was the following: KH2PO4 +Na2HPO4 x 12H2O + CgH12O6 x H2O in a:b:c ratio.
I) Effects of the above-mentioned medium on the spermatozoids (model/method of study)
a) survival period (minutes) of spermatozoa of the bull whose genital organs were affected with bacterial infection.
* (p < 0.01) b)~ Progressive sperm motility (%
c) Mode of spermatozoa movement in semen (native/frozen-thawed)
* (p < 0.01)
FIELD STUDIES OF THE MEDIUM (pH 6.75)
In order to demonstrate favorable effects of the medium on the sperm, three inseminations of the cows were performed in field conditions, with previous application of the medium. It was an adequate biological verification of the degree (%) of conception in cows.
Medium composition was the following: KH2PO4 +Na2HPO4 x 12H2O x C6Hi2O6 x H2O.
The medium (pH 6.75) was applied intracervically simultaneously or before insemination of the cows. Naturally, the cows were in estrus at the time. The insemination was performed lege artis, and thus bull semen dose used for insemination was mixed with the medium in the cervical canal, i.e., with estral mucus of the cows.
INSEMINATION OF CATTLE WITH APPLICATION OF THE STUDIED
MEDHJM (Ph 6.75) e) percentage (%) of fertility of the inseminated cows (gravidity, diagnosis per rectum)
* (ρ < 0.05)
Considering the obtained study results on the influence of the above-mentioned medium studied on the sperm (spermatozoa) of the infected bulls, the medium was proved to have highly favorable effects on the sex cell functions (p< 0.01), primarily on their viability, survival period, progressive movement and penetration capacity of the spermatozoa on the native and/or frozen semen. Otherwise, sperm-related parameters were notably less favorable in bulls with urogenital infections.
B) LABORATORY ANALYSES OF THE MEDIUM (pH 7.10) IN BULL SPERM AFFECTED BY URINARY INFECTION
Composition of the medium was the following: KH2PO4 +Na2HPO4 x 12H2O + C6H12O6 x H2O in a:b:c ratio
II) Effects of the above-mentioned medium on the spermatozoids (model/method of study)
a) survival period (minutes) of spermatozoa of the bull whose genital organs were affected with bacterial infection.
* (p < 0.01) b)- Progressive sperm motility (%)
c) Mode of spermatozoa movement in semen (native/frozen-thawed)
Before treatment After treatment ("ant-like" nervous - movement) prominently progressive - smooth of native spermatozoa of frozen sperm
d) penetration capacity - movement of spermatozoa through estral mucosa (mm/15 min.)
Before treatment After treatment (with medium) * " of of native spermatozoa (/15 min.) frozen spermatozoa (/15 min.)
25-30 45-75
* (p < 0.01)
FIELD STUDIES OF THE MEDIUM (pH 7.10)
The medium (pH 7.10) was also studied on the sperm of the breeding bulls using both native and frozen sperm, as well as the influence of the medium on the gravidity (conception) success rate (%) achieved in the inseminated cows.
Insemination of cattle with application of the studied medium (Ph 7.10)
e) percentage (%) of fertility of the inseminated cows (gravidity, diagnosis per rectum)
* (p < 0.05)
Based on the medium studies it may be concluded that the medium significantly influences cow insemination success rate owing to its favorable effects on the spermatozoa, that is their transport through the genital tract, viability, penetration capacity of the spermatozoa, i.e., their metabolism.
APPLICATION (ACTION)
The medium exerts its actions on the spermatozoa:
- It enhances the sperm metabolism, promotes poor motility of the spermatozoa resulting from infection of the genital organs - accessory glands of the male sex glands, improves their capacitation in the cervix uteri canal of the female animals, prolongs active penetration of the spermatozoa through the female genital tract, influences active progressive motility of spermatozoa, naturally (specifically) floating movement of the spermatozoa, saving at the same time their energy, has favorable influence on the so-called ant-like movement of the spermatozoa induced by genital infections, improves fertility of domestic male animals and humans upon coitus or insemination. The medium must be sterilized before administration! Shelf life of the medium is limitedl
Activity of the medium was also verified on the sperm of humans, rams and boars, when the medium was proved to have favorable effects on sperm viability and motility of the suitable male animals, prolonging their viability, i.e., their survival period in the external environment (in vitro).
Claims
1. The medium for improvement of sperm viability of domestic mammals and humans, whereas the medium is composed of the primary potassium phosphate, secondary sodium phosphate and monosaccharides in mix ratio: a) 44:36:20; b)45:30:25; c) 33.33:33.33:33.33.
2. The medium for improvement of viability of the spermatozoa of the domestic mammals (humans) according to the patent claim 1, whereas, the ratio is 33.33:33.33:33.33.
3. The medium for improvement of the viability of the spermatozoa of the domestic mammals (humans) according to the patent claims 1 and 2, whereas pH value ranges between 6.75 and 7.75.
4. The medium for improvement of the viability of the spermatozoa of the domestic mammals (humans) according to the patent claims 1- 3 whereas pH value is 7.10
5. The medium for improvement of the viability of the spermatozoa of the domestic mammals (humans) according to the patent claims 1- 4 whereas pH value is 7.20.
6. The medium for improvement of the viability of the spermatozoa of the domestic mammals (humans) according to the patent claims 1- 5 whereas monosaccharide is hexose.
7. The medium for improvement of the viability of the spermatozoa of the domestic mammals (humans) according to the patent claims 1- 6 whereas hexose is selected from the group comprising glucose, mannose, galactose and fructose.
8. The medium for improvement of the viability of the spermatozoa of the domestic mammals (humans) according to the patent claims 1- 7 whereas hexose is glucose.
9. The medium for improvement of the viability of the spermatozoa of the domestic mammals (humans) according to the patent claims 1- 7 whereas hexose is fructose.
10. Application of the medium for improvement of the viability of the spermatozoa of the domestic mammals (humans) according any of the above claims in the procedure of insemination and/or coitus of the domestic mammals (humans)
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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YUP-2005/0416 | 2005-05-30 | ||
YUP050416 | 2005-05-30 |
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WO2006130884A2 true WO2006130884A2 (en) | 2006-12-07 |
WO2006130884A3 WO2006130884A3 (en) | 2008-01-03 |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2326182A1 (en) * | 2007-09-29 | 2011-06-01 | Timothy James Williams | Composition and method to modify sperm fertility and female gender ratio in mammals |
WO2013086431A1 (en) * | 2011-12-09 | 2013-06-13 | The Curators Of The University Of Missouri | Inorganic pyrophosphate and uses thereof |
CN114317416A (en) * | 2022-01-14 | 2022-04-12 | 中国福利会国际和平妇幼保健院 | Application of mannose in improving sperm quality or/and fertilization rate |
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GB2102668A (en) * | 1981-07-29 | 1983-02-09 | Us Commerce | Turkey semen extender |
DD228439A1 (en) * | 1984-11-06 | 1985-10-16 | Inst Gefluegelwirtschaft Merbi | MEDIUM FOR THE DEHUMIDIFICATION, LIQUID AND FREEZER CONSERVATION OF PUTER SPERMA |
DD234222A1 (en) * | 1984-11-06 | 1986-03-26 | Inst Gefluegelwirtschaft Merbi | MEDIUM FOR THE DEGRADATION, LIQUID AND FREEZER CONSERVATION OF ERPELSPERMA |
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Title |
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DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; July 2001 (2001-07), WILLIAMS ANDREW C ET AL: "The role of glucose in supporting motility and capacitation in human spermatozoa" XP002452915 Database accession no. PREV200100381628 & JOURNAL OF ANDROLOGY, vol. 22, no. 4, July 2001 (2001-07), pages 680-695, ISSN: 0196-3635 * |
DATABASE CA [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; M.I.LOPATKO: "Tris phosphate medium for ram sperm" XP002452916 retrieved from STN-INTERNATIONAL Database accession no. 75:32591 & VISNIK SIL'S'KOGOSPODARS'KOI NAUKI, no. 4, 1971, pages 89-94, * |
DATABASE CA CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; J.STRZEZEK ET AL.: "Biochemical chnges in turkey semen during storage in a phosphate diluent" XP002452914 retrieved from STN-INTERNATIONAL Database accession no. 93:218329 & MEDYCYNA WETERYNARYJNA, vol. 36, no. 6, 1980, pages 356-359, * |
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Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2326182A1 (en) * | 2007-09-29 | 2011-06-01 | Timothy James Williams | Composition and method to modify sperm fertility and female gender ratio in mammals |
EP2326182A4 (en) * | 2007-09-29 | 2012-07-11 | Timothy James Williams | Composition and method to modify sperm fertility and female gender ratio in mammals |
WO2013086431A1 (en) * | 2011-12-09 | 2013-06-13 | The Curators Of The University Of Missouri | Inorganic pyrophosphate and uses thereof |
US8771934B2 (en) | 2011-12-09 | 2014-07-08 | The Curators Of The University Of Missouri | Inorganic pyrophosphate and uses thereof |
CN104394689A (en) * | 2011-12-09 | 2015-03-04 | 密苏里大学管理者 | Inorganic pyrophosphate and uses thereof |
EP2787810A4 (en) * | 2011-12-09 | 2015-05-27 | Univ Missouri | Inorganic pyrophosphate and uses thereof |
US10070889B2 (en) | 2011-12-09 | 2018-09-11 | The Curators Of The University Of Missouri | Inorganic pyrophosphate and uses thereof |
CN114317416A (en) * | 2022-01-14 | 2022-04-12 | 中国福利会国际和平妇幼保健院 | Application of mannose in improving sperm quality or/and fertilization rate |
CN114317416B (en) * | 2022-01-14 | 2023-12-15 | 中国福利会国际和平妇幼保健院 | Application of mannose in improving sperm quality and/or fertilization rate |
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